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Translocation of iron from lysosomes to mitochondria during ischemia predisposes to injury after reperfusion in rat hepatocytes.

ABSTRACT: The mitochondrial permeability transition (MPT) initiated by reactive oxygen species (ROS) plays an essential role in ischemia-reperfusion (IR) injury. Iron is a critical catalyst for ROS formation, and intracellular chelatable iron promotes oxidative injury-induced and MPT-dependent cell death in hepatocytes. Accordingly, our aim was to investigate the role of chelatable iron in IR-induced ROS generation, MPT formation, and cell death in primary rat hepatocytes. To simulate IR, overnight-cultured hepatocytes were incubated anoxically at pH 6.2 for 4h and reoxygenated at pH 7.4. Chelatable Fe(2+), ROS, and mitochondrial membrane potential were monitored by confocal fluorescence microscopy of calcein, chloromethyldichlorofluorescein, and tetramethylrhodamine methyl ester, respectively. Cell killing was assessed by propidium iodide fluorimetry. Ischemia caused progressive quenching of cytosolic calcein by more than 90%, signifying increased chelatable Fe(2+). Desferal and starch-desferal 1h before ischemia suppressed calcein quenching. Ischemia also induced quenching and dequenching of calcein loaded into mitochondria and lysosomes, respectively. Desferal, starch-desferal, and the inhibitor of the mitochondrial Ca(2+) uniporter (MCU), Ru360, suppressed mitochondrial calcein quenching during ischemia. Desferal, starch-desferal, and Ru360 before ischemia also decreased mitochondrial ROS formation, MPT opening, and cell killing after reperfusion. These results indicate that lysosomes release chelatable Fe(2+) during ischemia, which is taken up into mitochondria by MCU. Increased mitochondrial iron then predisposes to ROS-dependent MPT opening and cell killing after reperfusion.

SUBMITTER: Zhang X

PROVIDER: S-EPMC3932485 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Translocation of iron from lysosomes to mitochondria during ischemia predisposes to injury after reperfusion in rat hepatocytes.

Zhang Xun X Lemasters John J JJ

Free radical biology & medicine 20130509

The mitochondrial permeability transition (MPT) initiated by reactive oxygen species (ROS) plays an essential role in ischemia-reperfusion (IR) injury. Iron is a critical catalyst for ROS formation, and intracellular chelatable iron promotes oxidative injury-induced and MPT-dependent cell death in hepatocytes. Accordingly, our aim was to investigate the role of chelatable iron in IR-induced ROS generation, MPT formation, and cell death in primary rat hepatocytes. To simulate IR, overnight-cultur ...[more]

PMID: 23665427

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Project description:Introduction: Elevated levels of mitochondrial reactive oxygen species (ROS) contribute to the development of numerous cardiovascular diseases. TERT, the catalytic subunit of telomerase, has been shown to translocate to mitochondria to suppress ROS while promoting ATP production. Acute overexpression of TERT increases survival and decreases infarct size in a mouse model of myocardial infarct, while decreased telomerase activity predisposes to mitochondrial defects and heart failure. In the present study, we examined the role of TERT on cardiac structure and function under basal conditions and conditions of acute or prolonged stress in a novel rat model of TERT deficiency. Methods: Cardiac structure and function were evaluated via transthoracic echocardiogram. Langendorff preparations were used to test the effects of acute global ischemia reperfusion injury on cardiac function and infarction. Coronary flow and left ventricular pressure were measured during and after ischemia/reperfusion (I/R). Mitochondrial DNA integrity was measured by PCR and mitochondrial respiration was assessed in isolated mitochondria using an Oxygraph. Angiotensin II infusion was used as an established model of systemic stress. Results: No structural changes (echocardiogram) or coronary flow/left ventricle pressure (isolated hearts) were observed in TERT-/- rats at baseline; however, after I/R, coronary flow was significantly reduced in TERT-/- compared to wild type (WT) rats, while diastolic Left Ventricle Pressure was significantly elevated (n = 6 in each group; p < 0.05) in the TERT-/-. Interestingly, infarct size was less in TERT-/- rats compared to WT rats, while mitochondrial respiratory control index decreased and mitochondrial DNA lesions increased in TERT-/- compared to WT. Angiotensin II treatment did not alter cardiac structure or function; however, it augmented the infarct size significantly more in TERT-/- compared to the WT. Conclusion: Absence of TERT activity increases susceptibility to stress like cardiac injury. These results suggest a critical role of telomerase in chronic heart disease.

Project description:BackgroundRenal ischemia-reperfusion injury (IRI) is a major factor contributing to acute kidney injury and it is associated with a high morbidity and mortality if untreated. Renal IRI depletes cellular and tissue adenosine triphosphate (ATP), which compromises mitochondrial function, further exacerbating renal tubular injury. Currently, no treatment for IRI is available. This study investigates the protective role of treprostinil in improving mitochondria biogenesis and recovery during rat renal IRI.MethodsMale Sprague Dawley rats were randomly assigned to groups: control, sham, IRI-placebo or IRI-treprostinil and subjected to 45 min of bilateral renal ischemia followed by 1-72 h reperfusion. Placebo or treprostinil (100 ng/kg/min) was administered subcutaneously via an osmotic minipump.ResultsTreprostinil significantly reduced peak elevated serum creatinine (SCr) levels and accelerated normalization relative to IRI-placebo (p < 0.0001). Treatment with treprostinil also inhibited IRI-mediated renal apoptosis, mitochondrial oxidative injury (p < 0.05), and the release of cytochrome c (p < 0.01) vs. IRI-placebo. In addition, treprostinil preserved renal mitochondrial DNA copy number (p < 0.0001) and renal ATP levels (p < 0.05) to nearly those of sham-operated animals. Non-targeted semi-quantitative proteomics showed reduced levels of ATP synthase subunits in the IRI-placebo group which were restored to sham levels by treprostinil treatment (p < 0.05). Furthermore, treprostinil reduced renal IRI-induced upregulated Drp1 and pErk protein levels, and restored Sirt3 and Pgc-1α levels to baseline (p < 0.05).ConclusionsTreprostinil reduces mitochondrial-mediated renal apoptosis, inhibits mitochondria fission, and promotes mitochondria fusion, thereby accelerating mitochondrial recovery and protecting renal proximal tubules from renal IRI. These results support the clinical investigation of treprostinil as a viable therapy to reduce renal IRI.

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Translocation of iron from lysosomes to mitochondria during ischemia predisposes to injury after reperfusion in rat hepatocytes.

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Translocation of iron from lysosomes to mitochondria during ischemia predisposes to injury after reperfusion in rat hepatocytes.

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