Drp1/p53 interaction mediates p53 mitochondrial localization and dysfunction in septic cardiomyopathy.

BACKGROUND: Previous studies have implicated p53-dependent mitochondrial dysfunction in sepsis induced end organ injury, including sepsis-induced myocardial dysfunction (SIMD). However, the mechanisms behind p53 localization to the mitochondria have not been well established. Dynamin-related protein 1 (Drp1), a mediator of mitochondrial fission, may play a role in p53 mitochondrial localization. Here we examined the role of Drp1/p53 interaction in SIMD using in vitro and murine models of sepsis. METHODS: H9c2 cardiomyoblasts and BALB/c mice were exposed to lipopolysaccharide (LPS) to model sepsis phenotype. Pharmacologic inhibitors of Drp1 activation (ψDrp1) and of p53 mitochondrial binding (pifithrin µ, PFTµ) were utilized to assess interaction between Drp1 and p53, and the subsequent downstream impact on mitochondrial morphology and function, cardiomyocyte function, and sepsis phenotype. RESULTS: Both in vitro and murine models demonstrated an increase in physical Drp1/p53 interaction following LPS treatment, which was associated with increased p53 mitochondrial localization, and mitochondrial dysfunction. This Drp1/p53 interaction was inhibited by ΨDrp1, suggesting that this interaction is dependent on Drp1 activation. Treatment of H9c2 cells with either ΨDrp1 or PFTµ inhibited the LPS mediated localization of Drp1/p53 to the mitochondria, decreased oxidative stress, improved cellular respiration and ATP production. Similarly, treatment of BALB/c mice with either ΨDrp1 or PFTµ decreased LPS-mediated mitochondrial localization of p53, mitochondrial ROS in cardiac tissue, and subsequently improved cardiomyocyte contractile function and survival. CONCLUSION: Drp1/p53 interaction and mitochondrial localization is a key prodrome to mitochondrial damage in SIMD and inhibiting this interaction may serve as a therapeutic target.

Drp1/Fis1-Dependent Pathologic Fission and Associated Damaged Extracellular Mitochondria Contribute to Macrophage Dysfunction in Endotoxin Tolerance.

OBJECTIVES: Recent publications have shown that mitochondrial dynamics can govern the quality and quantity of extracellular mitochondria subsequently impacting immune phenotypes. This study aims to determine if pathologic mitochondrial fission mediated by Drp1/Fis1 interaction impacts extracellular mitochondrial content and macrophage function in sepsis-induced immunoparalysis. DESIGN: Laboratory investigation. SETTING: University laboratory. SUBJECTS: C57BL/6 and BALB/C mice. INTERVENTIONS: Using in vitro and murine models of endotoxin tolerance (ET), we evaluated changes in Drp1/Fis1-dependent pathologic fission and simultaneously measured the quantity and quality of extracellular mitochondria. Next, by priming mouse macrophages with isolated healthy mitochondria (MC) and damaged mitochondria, we determined if damaged extracellular mitochondria are capable of inducing tolerance to subsequent endotoxin challenge. Finally, we determined if inhibition of Drp1/Fis1-mediated pathologic fission abrogates release of damaged extracellular mitochondria and improves macrophage response to subsequent endotoxin challenge. MEASUREMENTS AND MAIN RESULTS: When compared with naïve macrophages (NMs), endotoxin-tolerant macrophages (ETM) demonstrated Drp1/Fis1-dependent mitochondrial dysfunction and higher levels of damaged extracellular mitochondria (Mitotracker-Green + events/50 µL: ETM = 2.42 × 106 ± 4,391 vs NM = 5.69 × 105 ± 2,478; p < 0.001). Exposure of NMs to damaged extracellular mitochondria (MH) induced cross-tolerance to subsequent endotoxin challenge, whereas MC had minimal effect (tumor necrosis factor [TNF]-α [pg/mL]: NM = 668 ± 3, NM + MH = 221 ± 15, and NM + Mc = 881 ± 15; p < 0.0001). Inhibiting Drp1/Fis1-dependent mitochondrial fission using heptapeptide (P110), a selective inhibitor of Drp1/Fis1 interaction, improved extracellular mitochondrial function (extracellular mitochondrial membrane potential, JC-1 [R/G] ETM = 7 ± 0.5 vs ETM + P110 = 19 ± 2.0; p < 0.001) and subsequently improved immune response in ETMs (TNF-α [pg/mL]; ETM = 149 ± 1 vs ETM + P110 = 1,150 ± 4; p < 0.0001). Similarly, P110-treated endotoxin tolerant mice had lower amounts of damaged extracellular mitochondria in plasma (represented by higher extracellular mitochondrial membrane potential, TMRM/MT-G: endotoxin tolerant [ET] = 0.04 ± 0.02 vs ET + P110 = 0.21 ± 0.02; p = 0.03) and improved immune response to subsequent endotoxin treatment as well as cecal ligation and puncture. CONCLUSIONS: Inhibition of Drp1/Fis1-dependent mitochondrial fragmentation improved macrophage function and immune response in both in vitro and in vivo models of ET. This benefit is mediated, at least in part, by decreasing the release of damaged extracellular mitochondria, which contributes to endotoxin cross-tolerance. Altogether, these data suggest that alterations in mitochondrial dynamics may play an important role in sepsis-induced immunoparalysis.

δPKC-Mediated DRP1 Phosphorylation Impacts Macrophage Mitochondrial Function and Inflammatory Response to Endotoxin.

BACKGROUND: Recent studies have demonstrated that alterations in mitochondrial dynamics can impact innate immune function. However, the upstream mechanisms that link mitochondrial dynamics to innate immune phenotypes have not been completely elucidated. This study asks if Protein Kinase C, subunit delta (δPKC)-mediated phosphorylation of dynamin-related protein 1 (Drp1), a key driver of mitochondrial fission, impacts macrophage pro-inflammatory response following bacterial-derived lipopolysaccharide (LPS) stimulation. METHODS: Using RAW 264.7 cells, bone marrow-derived macrophages from C57BL/6J mice, as well as human monocyte-derived macrophages, we first characterized changes in δPKC-mediated phosphorylation of Drp1 following LPS stimulation. Next, using rationally designed peptides that inhibit δPKC activation (δV1-1) and δPKC-Drp1 interaction (ψDrp1), we determined whether δPKC-mediated phosphorylation of Drp1 impacts LPS-induced changes in mitochondrial morphology, mitochondrial function, and inflammatory response. RESULTS: Our results demonstrated that δPKC-dependent Drp1 activation is associated with increased mitochondrial fission, impaired cellular respiration, and increased mitochondrial reactive oxygen species in LPS-treated macrophages. This is reversed using a rationally designed peptide that selectively inhibits δPKC phosphorylation of Drp1 (ψDrp1). Interestingly, limiting excessive mitochondrial fission using ψDrp1 reduced LPS-triggered pro-inflammatory response, including a decrease in NF-κB nuclear localization, decreased iNOS induction, and a reduction in pro-inflammatory cytokines (IL-1ß, TNFα, IL-6). CONCLUSION: These data suggest that inhibiting Drp1 phosphorylation by δPKC abates the excessive mitochondrial fragmentation and mitochondrial dysfunction that is seen following LPS treatment. Furthermore, these data suggest that limiting δPKC-dependent Drp1 activation decreases the pro-inflammatory response following LPS treatment. Altogether, δPKC-dependent Drp1 phosphorylation might be an upstream mechanistic link between alterations in mitochondrial dynamics and innate immune phenotypes, and may have therapeutic potential.

Cancer-associated fibroblasts downregulate type I interferon receptor to stimulate intratumoral stromagenesis.

Activation of cancer-associated fibroblasts (CAFs) and ensuing desmoplasia play an important role in the growth and progression of solid tumors. Here we demonstrate that, within colon and pancreatic ductal adenocarcinoma tumors, efficient stromagenesis relies on downregulation of the IFNAR1 chain of the type I interferon (IFN1) receptor. Expression of the fibroblast activation protein (FAP) and accumulation of the extracellular matrix (ECM) was notably impaired in tumors grown in the Ifnar1S526A (SA) knock-in mice, which are deficient in IFNAR1 downregulation. Primary fibroblasts from these mice exhibited elevated levels of Smad7, a negative regulator of the transforming growth factor-ß (TGFß) pathway. Knockdown of Smad7 alleviated deficient ECM production in SA fibroblasts in response to TGFß. Analysis of human colorectal cancers revealed an inverse correlation between IFNAR1 and FAP levels. Whereas growth of tumors in SA mice was stimulated by co-injection of wild type but not SA fibroblasts, genetic ablation of IFNAR1 in fibroblasts also accelerated tumor growth. We discuss how inactivation of IFNAR1 in CAFs acts to stimulate stromagenesis and tumor growth.

Mitochondrial dysfunction mediated through dynamin-related protein 1 (Drp1) propagates impairment in blood brain barrier in septic encephalopathy.

BACKGROUND: Out of the myriad of complications associated with septic shock, septic-associated encephalopathy (SAE) carries a significant risk of morbidity and mortality. Blood-brain-barrier (BBB) impairment, which subsequently leads to increased vascular permeability, has been associated with neuronal injury in sepsis. Thus, preventing BBB damage is an attractive therapeutic target. Mitochondrial dysfunction is an important contributor of sepsis-induced multi-organ system failure. More recently, mitochondrial dysfunction in endothelial cells has been implicated in mediating BBB failure in stroke, multiple sclerosis and in other neuroinflammatory disorders. Here, we focused on Drp1-mediated mitochondrial dysfunction in endothelial cells as a potential target to prevent BBB failure in sepsis. METHODS: We used lipopolysaccharide (LPS) to induce inflammation and BBB disruption in a cell culture as well as in murine model of sepsis. BBB disruption was assessed by measuring levels of key tight-junction proteins. Brain cytokines levels, oxidative stress markers, and activity of mitochondrial complexes were measured using biochemical assays. Astrocyte and microglial activation were measured using immunoblotting and qPCR. Transwell cultures of brain microvascular endothelial cells co-cultured with astrocytes were used to assess the effect of LPS on expression of tight-junction proteins, mitochondrial function, and permeability to fluorescein isothiocyanate (FITC) dextran. Finally, primary neuronal cultures exposed to LPS were assessed for mitochondrial dysfunction. RESULTS: LPS induced a strong brain inflammatory response and oxidative stress in mice which was associated with increased Drp1 activation and mitochondrial localization. Particularly, Drp1-(Fission 1) Fis1-mediated oxidative stress also led to an increase in expression of vascular permeability regulators in the septic mice. Similarly, mitochondrial defects mediated via Drp1-Fis1 interaction in primary microvascular endothelial cells were associated with increased BBB permeability and loss of tight-junctions after acute LPS injury. P110, an inhibitor of Drp1-Fis1 interaction, abrogated these defects, thus indicating a critical role for this interaction in mediating sepsis-induced brain dysfunction. Finally, LPS mediated a direct toxic effect on primary cortical neurons, which was abolished by P110 treatment. CONCLUSIONS: LPS-induced impairment of BBB appears to be dependent on Drp1-Fis1-mediated mitochondrial dysfunction. Inhibition of mitochondrial dysfunction with P110 may have potential therapeutic significance in septic encephalopathy.

Drp1/Fis1 interaction mediates mitochondrial dysfunction in septic cardiomyopathy.

Mitochondrial dysfunction is a key contributor to septic cardiomyopathy. Although recent literature implicates dynamin related protein 1 (Drp1) and its mitochondrial adaptor fission 1 (Fis1) in the development of pathologic fission and mitochondrial failure in neurodegenerative disease, little is known about the role of Drp1/Fis1 interaction in the context of sepsis-induced cardiomyopathy. Our study tests the hypothesis that Drp1/Fis1 interaction is a major driver of sepsis-mediated pathologic fission, leading to mitochondrial dysfunction in the heart. METHODS: H9C2 cardiomyocytes were treated with lipopolysaccharide (LPS) to evaluate changes in mitochondrial membrane potential, oxidative stress, cellular respiration, and mitochondrial morphology. Balb/c mice were treated with LPS, cardiac function was measured by echocardiogaphy, and mitochondrial morphology determined by electron microscopy (EM). Drp1/Fis1 interaction was inhibited by P110 to determine whether limiting mitochondrial fission can reduce LPS-induced oxidative stress and cardiac dysfunction. RESULTS: LPS-treated H9C2 cardiomyocytes demonstrated a decrease in mitochondrial respiration followed by an increase in mitochondrial oxidative stress and a reduction in membrane potential. Inhibition of Drp1/Fis1 interaction with P110 attenuated LPS-mediated cellular oxidative stress and preserved membrane potential. In vivo, cardiac dysfunction in LPS-treated mice was associated with increased mitochondrial fragmentation. Treatment with P110 reduced cardiac mitochondrial fragmentation, prevented decline in cardiac function, and reduced mortality. CONCLUSIONS: Sepsis decreases cardiac mitochondrial respiration and membrane potential while increasing oxidative stress and inducing pathologic fission. Treatment with P110 was protective in both in vitro and in vivo models of septic cardiomyopathy, suggesting a key role of Drp1/Fis1 interaction, and a potential target to reduce its morbidity and mortality.