Introduction Mitochondria supply cellular energy and are key regulators of intrinsic cell death and consequently affect longevity. The nematode Caenorhabditis elegans is frequently used for lifespan assays. Using paraquat (PQ) as a generator of reactive oxygen species, we here describe its effects on the acceleration of aging and the associated dysfunctions at the level of mitochondria. Methods Nematodes were incubated with various concentrations of paraquat in a heat-stress resistance assay (37°C) using nucleic staining. The most effective concentration was validated under physiological conditions, and chemotaxis was assayed. Mitochondrial membrane potential (ΔΨm) was measured using rhodamine 123, and activity of respiratory chain complexes determined using a Clark-type electrode in isolated mitochondria. Energetic metabolites in the form of pyruvate, lactate, and ATP were determined using commercial kits. Mitochondrial integrity and structure was investigated using transmission electron microscopy. Live imaging after staining with fluorescent dyes was used to measure mitochondrial and cytosolic ROS. Expression of longevity- and mitogenesis-related genes were evaluated using qRT-PCR. Results PQ (5 mM) significantly increased ROS formation in nematodes and reduced the chemotaxis, the physiological lifespan, and the survival in assays for heat-stress resistance. The number of fragmented mitochondria significantly increased. The ∆Ψm, the activities of complexes I-IV of the mitochondrial respiratory chain, and the levels of pyruvate and lactate were significantly reduced, whereas ATP production was not affected. Transcript levels of genetic marker genes, atfs-1, atp-2, skn-1, and sir-2.1, were significantly upregulated after PQ incubation, which implicates a close connection between mitochondrial dysfunction and oxidative stress response. Expression levels of aak-2 and daf-16 were unchanged. Conclusion Using paraquat as a stressor, we here describe the association of oxidative stress, restricted energy metabolism, and reduced stress resistance and longevity in the nematode Caenorhabditis elegans making it a readily accessible in vivo model for mitochondrial dysfunction.
Caenorhabditis elegans is widely used for aging studies. 5-Fluoro-2´-deoxyuridine (FUdR) is commonly used to control offspring. While larvae are stopped from further development, also mitochondrial DNA and function may be affected. Since mitochondria and longevity are closely related, the use of FUdR may falsify possible studies. PX627, an auxin inducible infertility strain to control offspring, allows mitochondrial investigations during senescence without FUdR toxicity. Longevity and health parameters were assessed in 2-and 10-day old nematodes wild-type N2 and PX627 treated with FUdR or auxin, respectively. Mitochondrial membrane potential, energetic metabolites and reactive oxygen species levels, were determined. mRNA expression levels of key genes involved were quantified using quantitative real-time PCR. FUdR significantly increased lifespan and health parameters, as well as, mitochondrial function compared to untreated controls and auxin treated PX627. Although a decrease in all parameters could be observed in aged nematodes, this was less severe after FUdR exposure. Glycolysis was significantly up-regulated in aged PX627 compared to N2. Expression levels of daf-16, sir-2.1, aak-2, skn-1, atp-2 and atfs-1 were regulated accordingly. Hence, auxin in PX627 might be a good alternative to control progeny, for mitochondrial-and longevity-related investigations in nematodes.
Die Bestimmung der fraktionellen Flussreserve (FFR) unter Bedingungen der maximalen Hyper?mie ? induziert mit Adenosin ? im gemessenen koronaren Versorgungsgebiet ist eine einfache und valide Methode zur h?modynamischen Quantifizierung von Koronarstenosen und damit derzeit der Goldstandard zur Therapie-Entscheidung f?r den invasiv t?tigen Kardiologen. Ein Wert?0,80 stellt eine klare, auch prognostische, Indikation f?r eine Intervention dar. Bei Werten>0,80 sollte, unter prognostischen Aspekten, auf eine Revaskularisation verzichtet werden. Die Evaluation von Stenosen mit der ? Adenosin-freien ? instantaneous wave free ratio (iFR) ist ebenfalls m?glich bei Patienten, die aufgrund von Kontraindikationen kein Adenosin erhalten d?rfen. Aufgrund der geringen diagnostischen Genauigkeit in den aktuellen Studien ist ihr routinem??iger Einsatz zurzeit aber limitiert.
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