Tubulin ligands suggest a microtubule-NADPH oxidase relationship in postischemic cardiomyocytes.

Alterations of the microtubule network, which is involved in many vital processes, occur in several pathological conditions, such as cardiac ischemia. However, the connection between the microtubule assembly state and the factors affecting myocardial reperfusion injury, especially oxidative stress, is unknown. We aimed thus to study the effects of different tubulin ligands on the changes in the microtubule network and in several markers of cell injury and oxidative activity in cardiac muscle cells submitted to a reversible substrate-free, hypoxia-reoxygenation model of ischemia-reperfusion. The microtubule network was visualized by immunocytochemistry. Cell injury was evaluated via lactate dehydrogenase release and the mitochondrial function by the MTT test. Superoxide production was detected using dihydroethidium. The activity of NADPH oxidase and mRNA subunit expression were investigated. The microtubule disassembly induced by simulated ischemia was reversed by placing cardiomyocytes under normoxic conditions. This post-"ischemic" restoration of microtubule assembly was modulated by microtubule stabilizers (taxol: paclitaxel) and by microtubule disrupting drugs (nocodazole, colchicine). In addition, nocodazole decreased superoxide anion production as well as NADPH oxidase activity and mRNA expression of the NADPH oxidase subunit p22phox. These results demonstrated that the "ischemia"-induced microtubule network alteration is reversible and suggest a possible relationship between "reperfusion"-induced reassembly of microtubules and free radical generation in post-"ischemic" cardiomyocytes.

Dependence on the phospholipid polyunsaturated fatty acids of the oxidative injury of isolated cardiomyocytes.

We investigated the influence of PUFA in phospholipids (PL) on the functional characteristics of cultured cardiomyocytes (CM) in basal conditions and during free radical (FR) stress provoked either by the xanthine/xanthine oxidase (X/XO) system or by a (9Z, 11E, 13 (S), 15Z)-13-hydroperoxyoctadecatrienoic acid (13-HpOTrE). CM were grown in media containing either n - 3 (eicosapentaenoic acid, EPA, and docosahexaenoic acid, DHA) or n - 6 (arachidonic acid, AA). These two groups of CM displayed different PUFA n - 6/n - 3 ratio in PL. However, their basal electromechanical characteristics were similar. The X/XO system drastically altered CM functions, without difference between the two groups of CM. 13-HpOTrE caused a moderate and reversible depression in action potential parameters, which was dependent upon the PL PUFA, since the n - 3-enriched CM exhibited an earlier functional depression but faster recovery. Thus, the peroxidative damage of CM depended on a cross relationship between FR species and the PL PUFA composition.

Direct, pleiotropic protective effect of cyclosporin A against simulated ischemia-induced injury in isolated cardiomyocytes.

Cyclosporin A is an immunosuppressor that prolongs graft survival but its use is limited by cardiotoxicity. The effects of cyclosporin A on several functional and biological characteristics were thus evaluated in rat cardiomyocytes in normal conditions and in a substrate-free, hypoxia-reoxygenation model of ischemia-reperfusion. Cyclosporin A (100 and 1000 ng/ml) did not induce cardiocytotoxicity in basal conditions. Simulated ischemia gradually decreased and then blocked the spontaneous electromechanical activity. Cyclosporin A at 100 and 1000 ng/ml permitted the maintenance of electromechanical functions that were abolished in control cells. Cyclosporin A also improved the post-"ischemic" functional recovery. Cyclosporin A reduced the "ischemia"-induced lactate dehydrogenase and troponine I releases and the successive rises in heat shock protein mRNA observed after "ischemia" and reoxygenation. Moreover, cyclosporin A improved the resumption of the mitochondrial function. To conclude, cyclosporin A displayed a direct, pleiotropic protection of isolated cardiomyocytes against physiological, metabolic, structural and stress signaling changes induced by ischemia-reperfusion mimicked in vitro.

Involvement of microtubules in the tolerance of cardiomyocytes to cold ischemia-reperfusion.

Before transplantation, the heart graft is preserved by the use of cold storage in order to limit ischemia-reperfusion stress. However, sustained exposure to low temperature may induce myocardial ultrastructural damage, particularly microtubules (MT) disruption. Previous data suggested that tubulin-binding agents are able to attenuate cold-induced cytoskeleton alterations. Thus, the aim of the present work was to study the influence of docetaxel (DX, a tubulin-binding taxane) on the effects of deep hypothermia (4 degrees C) and of simulated cold ischemia-reperfusion on the MT network and oxidative stress of cardiomyocyte (CM) in monolayer cultures prepared from newborn rat ventricles. The MT network was explored by immunocytochemistry and Western-blotting, the cell stress by tetrazolium dye assay (MTT) and lactate dehydrogenase (LDH) release, and the superoxide production by the dihydroethidium probe (DHE). The MT assembly remained stable after 4 and 8 h of hypothermia. Tubulin acetylation was promoted in CM subjected to 4-h hypothermia. Low temperature reduced the mitochondrial function and increased the basal LDH release. The cold ischemia during 4 and 8 h preserved MT network. Docetaxel promoted MT polymerization and tubulin acetylation in basal and in cold conditions. This drug decreased the release of LDH induced by cold ischemia. Moreover, hypothermia (4 h) significantly raised the anion superoxide production. Docetaxel decreased this oxidative stress in the control CM and in CM submitted to 4 h of hypothermia. These data demonstrated that stabilizing MT with DX exerted a protective effect on CM subjected to hypothermia and to cold ischemia-reperfusion. Tubulin-ligands should be thus considered to improve the tolerance of the heart graft toward stressing conservative conditions.

Substrate dependence of the postischemic cardiomyocyte recovery: dissociation between functional, metabolic and injury markers.

Defining the substrate that influences the most favourably the myocardial post-ischemic recovery is subject of debates, due to dissociation between functional and biochemical benefits. Hence, we studied the effects of either glucose or different fatty acids on the functional and metabolic recovery of post-ischemic cardiomyocytes in a substrate-free hypoxia model of simulated ischemia-reperfusion. Rat cardiomyocytes were submitted to a 2.5 h simulated ischemia followed by a 2 h reoxygenation without substrate (control), or with either glucose, octanoic acid, oleic acid, or elaidic acid. During simulated ischemia, electromechanical function gradually disappeared while the cellular viability and mitochondrial function declined. During control simulated reperfusion, cardiomyocytes recovered near normal function but a significant reduction in the action potential amplitude and rate persisted. The addition of glucose or oleic acid during simulated reperfusion promoted a faster, better and sustain functional recovery. Amongst the fatty acids, the functional recovery was slower with elaidic and octanoic acids as compared with oleic acid. The mitochondrial function was better improved during simulated reperfusion with glucose than with the tested fatty acids, among which elaidic acid was the less unfavourable. Paradoxically, the addition of whichever substrate during simulated reperfusion tended to worsen the cellular viability. Thus, cardiomyocytes recovery strongly relies on the characteristics of the substrate supplied at the onset of simulated reperfusion: glucidic or lipidic nature, chain-length, insaturation degree. Moreover, these data suggest that defining the appropriateness of a given substrate for the post-ischemic cardiomyocyte recovery is closely related to the functional and the biological endpoints in consideration.