Epoxyeicosatrienoic acids pretreatment improves amyloid ß-induced mitochondrial dysfunction in cultured rat hippocampal astrocytes.

Amyloid-ß (Aß) has long been implicated as a causative protein in Alzheimer's disease. Cellular Aß accumulation is toxic and causes mitochondrial dysfunction, which precedes clinical symptoms of Alzheimer's disease pathology. In the present study, we explored the possible use of epoxyeicosatrienoic acids (EETs), epoxide metabolites of arachidonic acid, as therapeutic target against Aß-induced mitochondrial impairment using cultured neonatal hippocampal astrocytes. Inhibition of endogenous EET production by a selective epoxygenase inhibitor, MS-PPOH, caused a greater reduction in mitochondrial membrane potential in the presence of Aß (1, 10 µM) exposure versus absence of Aß. MS-PPOH preincubation also aggravated Aß-induced mitochondrial fragmentation. Preincubation of the cells with either 14,15- or 11,12-EET prevented this mitochondrial depolarization and fragmentation. EET pretreatment also further improved the reduction observed in mitochondrial oxygen consumption in the presence of Aß. Preincubation of the cells with EETs significantly improved cellular respiration under basal condition and in the presence of the protonophore, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). The uncoupling of ATP synthase from the electron transfer chain that occurred in Aß-treated cells was also prevented by preincubation with EETs. Lastly, cellular reactive oxygen species production, a hallmark of Aß toxicity, also showed significant reduction in the presence of EETs. We have previously shown that Aß reduces EET synthesis in rat brain homogenates and cultured hippocampal astrocytes and neurons (Sarkar P, Narayanan J, Harder DR. Differential effect of amyloid beta on the cytochrome P450 epoxygenase activity in rat brain. Neuroscience 194: 241-249, 2011). We conclude that reduction of endogenous EETs may be one of the mechanisms through which Aß inflicts toxicity and thus supplementing the cells with exogenous EETs improves mitochondrial dynamics and prevents metabolic impairment.

Mitochondrial approaches to protect against cardiac ischemia and reperfusion injury.

The mitochondrion is a vital component in cellular energy metabolism and intracellular signaling processes. Mitochondria are involved in a myriad of complex signaling cascades regulating cell death vs. survival. Importantly, mitochondrial dysfunction and the resulting oxidative and nitrosative stress are central in the pathogenesis of numerous human maladies including cardiovascular diseases, neurodegenerative diseases, diabetes, and retinal diseases, many of which are related. This review will examine the emerging understanding of the role of mitochondria in the etiology and progression of cardiovascular diseases and will explore potential therapeutic benefits of targeting the organelle in attenuating the disease process. Indeed, recent advances in mitochondrial biology have led to selective targeting of drugs designed to modulate or manipulate mitochondrial function, to the use of light therapy directed to the mitochondrial function, and to modification of the mitochondrial genome for potential therapeutic benefit. The approach to rationally treat mitochondrial dysfunction could lead to more effective interventions in cardiovascular diseases that to date have remained elusive. The central premise of this review is that if mitochondrial abnormalities contribute to the etiology of cardiovascular diseases (e.g., ischemic heart disease), alleviating the mitochondrial dysfunction will contribute to mitigating the severity or progression of the disease. To this end, this review will provide an overview of our current understanding of mitochondria function in cardiovascular diseases as well as the potential role for targeting mitochondria with potential drugs or other interventions that lead to protection against cell injury.

Isoflurane preconditioning elicits competent endogenous mechanisms of protection from oxidative stress in cardiomyocytes derived from human embryonic stem cells.

BACKGROUND: Human embryonic stem cell (hESC)-derived cardiomyocytes potentially represent a powerful experimental model complementary to myocardium obtained from patients that is relatively inaccessible for research purposes. We tested whether anesthetic-induced preconditioning (APC) with isoflurane elicits competent protective mechanisms in hESC-derived cardiomyocytes against oxidative stress to be used as a model of human cardiomyocytes for studying preconditioning. METHODS: H1 hESC cell line was differentiated into cardiomyocytes using growth factors activin A and bone morphogenetic protein-4. Living ventricular hESC-derived cardiomyocytes were identified using a lentiviral vector expressing a reporter gene (enhanced green fluorescent protein) driven by a cardiac-specific human myosin light chain-2v promoter. Mitochondrial membrane potential, reactive oxygen species production, opening of mitochondrial permeability transition pore, and survival of hESC-derived cardiomyocytes were assessed using confocal microscopy. Oxygen consumption was measured in contracting cell clusters. RESULTS: Differentiation yielded a high percentage (∼85%) of cardiomyocytes in beating clusters that were positive for cardiac-specific markers and exhibited action potentials resembling those of mature cardiomyocytes. Isoflurane depolarized mitochondria, attenuated oxygen consumption, and stimulated generation of reactive oxygen species. APC protected these cells from oxidative stress-induced death and delayed mitochondrial permeability transition pore opening. CONCLUSIONS: APC elicits competent protective mechanisms against oxidative stress in hESC-derived cardiomyocytes, suggesting the feasibility to use these cells as a model of human cardiomyocytes for studying APC and potentially other treatments/diseases. Our differentiation protocol is very efficient and yields a high percentage of cardiomyocytes. These results also suggest a promising ability of APC to protect and improve engraftment of hESC-derived cardiomyocytes into the ischemic heart.

Xenon preconditioning: the role of prosurvival signaling, mitochondrial permeability transition and bioenergetics in rats.

BACKGROUND: Similar to volatile anesthetics, the anesthetic noble gas xenon protects the heart from ischemia/reperfusion injury, but the mechanisms responsible for this phenomenon are not fully understood. We tested the hypothesis that xenon-induced cardioprotection is mediated by prosurvival signaling kinases that target mitochondria. METHODS: Male Wistar rats instrumented for hemodynamic measurements were subjected to a 30 min left anterior descending coronary artery occlusion and 2 h reperfusion. Rats were randomly assigned to receive 70% nitrogen/30% oxygen (control) or three 5-min cycles of 70% xenon/30% oxygen interspersed with the oxygen/nitrogen mixture administered for 5 min followed by a 15 min memory period. Myocardial infarct size was measured using triphenyltetrazolium staining. Additional hearts from control and xenon-pretreated rats were excised for Western blotting of Akt and glycogen synthase kinase 3 beta (GSK-3beta) phosphorylation and isolation of mitochondria. Mitochondrial oxygen consumption before and after hypoxia/reoxygenation and mitochondrial permeability transition pore opening were determined. RESULTS: Xenon significantly (P < 0.05) reduced myocardial infarct size compared with control (32 +/- 4 and 59% +/- 4% of the left ventricular area at risk; mean +/- sd) and enhanced phosphorylation of Akt and GSK-3beta. Xenon pretreatment preserved state 3 respiration of isolated mitochondria compared with the results obtained in the absence of the gas. The Ca(2+) concentration required to induce mitochondrial membrane depolarization was larger in the presence compared with the absence of xenon pretreatment (78 +/- 17 and 56 +/- 17 microM, respectively). The phosphoinositol-3-kinase-kinase inhibitor wortmannin blocked the effect of xenon on infarct size and respiration. CONCLUSIONS: These results indicate that xenon preconditioning reduces myocardial infarct size, phosphorylates Akt, and GSK-3beta, preserves mitochondrial function, and inhibits Ca(2+)-induced mitochondrial permeability transition pore opening. These data suggest that xenon-induced cardioprotection occurs because of activation of prosurvival signaling that targets mitochondria and renders them less vulnerable to ischemia-reperfusion injury.