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Aim: In most vertebrates, oxygen deprivation and subsequent re‐oxygenation are associated with mitochondrial impairment and excess production of reactive oxygen species (ROS) like hydrogen peroxide (H2O2). This in turn triggers a cascade of cell‐damaging events in a temperature‐dependent manner. The crucian carp (Carassius carassius) is one of few vertebrates that survives months without oxygen at cold temperatures and overcomes oxidative damage during re‐oxygenation periods. Mitochondria of this anoxia‐tolerant species therefore serve as an excellent model in translational research to study adaptation and resilience to low oxygen conditions and thermal variability. Methods: Here, we used high‐resolution respirometry on isolated mitochondria from hearts of crucian carp and the anoxia‐intolerant mouse (Mus musculus), at 37 and 8°C; two temperatures relevant for transplantation medicine (i.e., graft preservation and subsequent rewarming). Results: We find: (1) a striking difference in H2O2 release between the two species at 37°C despite comparable mitochondrial efficiency and capacity, (2) a massive H2O2 release after inhibition of complex V in mouse at 37°C that is absent in crucian carp, and prevented in mouse by incubation at 8°C or uncoupling with a protonophore at 37°C, and (3) indications that differences in mitochondrial complex I and II capacity and thermal sensitivity influence the release of mitochondrial H2O2 relative to respiration. Conclusion: Our findings provide comparative insights into a spectrum of mitochondrial adaptations in vertebrates and the importance of thermal variability. Furthermore, the species‐ and temperature‐related changes associated with mitochondria highlighted in this study may help identify mitochondria‐based targets for translational medicine. [ABSTRACT FROM AUTHOR]

Cardiac cell death after myocardial infarction release endogenous structures termed damage‐associated molecular patterns (DAMPs) that trigger the innate immune system and initiate a sterile inflammation in the myocardium. Cardiomyocytes are energy demanding cells and 30% of their volume are mitochondria. Mitochondria are evolutionary endosymbionts originating from bacteria containing molecular patterns similar to bacteria, termed mitochondrial DAMPs (mDAMPs). Consequently, mitochondrial debris may be particularly immunogenic and damaging. However, the role of mDAMPs in myocardial infarction is not clarified. Identifying the most harmful mDAMPs and inhibiting their early inflammatory signaling may reduce infarct size and the risk of developing post‐infarct heart failure. The focus of this review is the role of mDAMPs in the immediate pro‐inflammatory phase after myocardial infarction before arrival of immune cells in the myocardium. We discuss different mDAMPs, their role in physiology and present knowledge regarding their role in the inflammatory response of acute myocardial infarction. [ABSTRACT FROM AUTHOR]

Background and Purpose: Cellular debris causes sterile inflammation after myocardial infarction. Mitochondria constitute about 30 percent of the human heart. Mitochondrial DNA (mtDNA) is a damage-associated-molecular-pattern that induce injurious sterile inflammation. Little is known about mtDNA's inflammatory signalling pathways in cardiomyocytes and how mtDNA is internalized to associate with its putative receptor, toll-like receptor 9 (TLR9).Experimental Approach: We hypothesized that mtDNA can be internalized in cardiomyocytes and induce an inflammatory response. Adult mouse cardiomyocytes were exposed to hypoxia-reoxygenation and extracellular DNA. Microscale thermophoresis was used to demonstrate binding between nucleolin and DNA.Key Results: Expression of the pro-inflammatory cytokines IL-1β and TNFα were upregulated by mtDNA, but not by nuclear DNA (nDNA), in cardiomyocytes exposed to hypoxia-reoxygenation. Blocking the RNA/DNA binding protein nucleolin with midkine reduced expression of IL-1β/TNFα and the nucleolin inhibitor AS1411 reduced interleukin-6 release in adult mouse cardiomyocytes. mtDNA bound 10-fold stronger than nDNA to nucleolin. In HEK293-NF-κB reporter cells, mtDNA induced NF-κB activity in normoxia, while CpG-DNA and hypoxia-reoxygenation, synergistically induced TLR9-dependent NF-κB activity. Protein expression of nucleolin was found in the plasma membrane of cardiomyocytes and inhibition of nucleolin with midkine inhibited cellular uptake of CpG-DNA. Inhibition of endocytosis did not reduce CpG-DNA uptake in cardiomyocytes.Conclusion and Implications: mtDNA, but not nDNA, induce an inflammatory response in mouse cardiomyocytes during hypoxia-reoxygenation. In cardiomyocytes, nucleolin is expressed on the membrane and blocking nucleolin reduce inflammation. Nucleolin might be a therapeutic target to prevent uptake of immunogenic DNA and reduce inflammation.Linked Articles: This article is part of a themed section on Mitochondrial Pharmacology: Featured Mechanisms and Approaches for Therapy Translation. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.22/issuetoc. [ABSTRACT FROM AUTHOR]

Background: IL-1β is a highly potent pro-inflammatory cytokine and its secretion is tightly regulated. Inactive pro-IL-1β is transcribed in response to innate immune receptors activating NFκB. If tissue damage occurs, danger signals released from necrotic cells, such as ATP, can activate NLRP3-inflammasomes (multiprotein complexes consisting of NLRP3, ASC, and active caspase-1) which cleaves and activates pro-IL-1β. NLRP3 activation also depends on NEK7 and mitochondrial ROS-production. Thus, IL-1β secretion may be regulated at the level of each involved component. We have previously shown that NLRP3-dependent IL-1β release can be induced in cardiac fibroblasts by pro-inflammatory stimuli. However, anti-inflammatory mechanisms targeting IL-1β release in cardiac cells have not been investigated. mTOR is a key regulator of protein metabolism, including autophagy and proteasome activity. In this study we explored whether autophagy or proteasomal degradation are regulators of NLRP3 inflammasome activation and IL-1β release from cardiac fibroblasts. Methods and Results: Serum starvation selectively reduced LPS/ATP-induced IL-1β secretion from cardiac fibroblasts. However, no other inflammasome components, nor mitochondrial mass, were affected. The mTOR inhibitor rapamycin restored pro-IL-1β protein levels as well as LPS/ATP-induced IL-1β release from serum starved cells. However, neither serum starvation nor rapamycin induced autophagy in cardiac fibroblasts. Conversely, chloroquine and bafilomycin A (inhibitors of autophagy) and betulinic acid (a proteasome activator) effectively reduced LPS-induced pro-IL-1β protein levels. Key findings were reinvestigated in human monocyte-derived macrophages. Conclusion: In cardiac fibroblasts, mTOR inhibition selectively favors pro-IL-1β synthesis while proteasomal degradation and not autophagy is the major catabolic anti-inflammatory mechanism for degradation of this cytokine. [ABSTRACT FROM AUTHOR]

Crucian carp (Carassius carassius) survive without oxygen for several months, but it is unknown whether they are able to protect themselves from cell death normally caused by the absence, and particularly return, of oxygen. Here, we quantified cell death in brain tissue from crucian carp exposed to anoxia and re-oxygenation using the terminal deoxy-nucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, and cell proliferation by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) as well as PCNA mRNA expression. We also measured mRNA and protein expression of the apoptosis executer protease caspase 3, in laboratory fish exposed to anoxia and re-oxygenation and fish exposed to seasonal anoxia and reoxygenation in their natural habitat over the year. Finally, a behavioural experiment was used to assess the ability to learn and remember how to navigate in a maze to find food, before and after exposure to anoxia and re-oxygenation. The number of TUNEL-positive cells in the telencephalon increased after 1 day of re-oxygenation following 7 days of anoxia, indicating increased cell death. However, therewere no consistent changes in whole-brain expression of caspase 3 in either laboratory-exposed or naturally exposed fish, indicating that cell death might occur via caspase-independent pathways or necrosis. Re-oxygenated crucian carp appeared to have lost the memory of how to navigate in a maze (learnt prior to anoxia exposure), while the ability to learn remained intact. PCNA mRNA was elevated after re-oxygenation, indicating increased neurogenesis. We conclude that anoxia tolerance involves not only protection from damage but also repair after re-oxygenation. [ABSTRACT FROM AUTHOR]

Introduction: The heart is a "mitochondrion-dense" organ, and as much as 30% of the volume of the cardiomyocyte is occupied by mitochondria. Mitochondria are both the target and origin of many of the pathogenic pathways in the development of heart failure, including metabolic remodeling and increased oxidative stress. Recent evidence suggests that mitochondria are the main source of inflammasome-activating short-lived reactive oxygen species (ROS), and as such may constitute the signal-integrating organelle for NLRP3 inflammasome activation. Methods: Adult mouse cardiomyocytes were isolated from normal C57Bl6 (WT) or mice deficient in NLRP3. Cardiomyocytes were incubated with the mitochondrial uncoupler (carbonyl cyanide m -chlorophenylhydrazone; CCCP) for 24 h. Cell viability was visualized by Annexin V (apoptosis) and propidium iodide (necrosis) staining. The number of positive cells was calculated. Cardiomyocytes were also loaded with the dye tetramethylrhodamine methyl ester (TMRM) and 20 min later subjected to ROS injury evoked by laser confocal microscopy. The time to membrane depolarization was measured and compared with control cells. Mitochondria were isolated from heart tissue after ex vivo ischemia reperfusion (Langendorff technique) and oxygen consumption by high-resolution respirometry was measured. NLRP3 protein was visualized using antibody staining and confocal microscopy. Results: Adult NLRP3–/– cardiomyocytes had reduced CCCP-induced cell death as compared to WT cardiomyocytes, as evident by decreased number of Annexin V and propidium iodide positive cells, Moreover, NLRP3-/- cells had slower mitochondrial membrane depolarization evaluated in TMRM loaded cardiomyocytes during exposure to reactive ROS induced by laser confocal microscopy, indicating protection of mitochondrial function. WT mitochondria from heart had reduced oxygen consumption after ischemia reperfusion compared to NLRP3–/– mitochondria demonstrating mitochondrial respiration impairment. Perinuclear NLRP3 protein was detected by laser confocal microscopy in the cardiomyocytes. Conclusions: NLRP3 in adult cardiac myocytes may play a role in regulating mitochondrial and cellular viability. If this is an effect dependent on inflammasome activity remains to be determined, but our results are compatible with the notion that ROS generated by mitochondria having reduced membrane potential can lead to NLRP3 inflammasome activation in cardiomyocytes and further to cardiac dysfunction. [ABSTRACT FROM AUTHOR]