Cardioprotection induced by remote ischemic preconditioning preserves the mitochondrial respiratory function in acute diabetic myocardium.

A 2×2 factorial design was used to evaluate possible preservation of mitochondrial functions in two cardioprotective experimental models, remote ischemic preconditioning and streptozotocin-induced diabetes mellitus, and their interaction during ischemia/reperfusion injury (I/R) of the heart. Male Wistar rats were randomly allocated into four groups: control (C), streptozotocin-induced diabetic (DM), preconditioned (RPC) and preconditioned streptozotocin-induced diabetic (DM+RPC). RPC was conducted by 3 cycles of 5-min hind-limb ischemia and 5-min reperfusion. DM was induced by a single dose of 65 mg/kg streptozotocin. Isolated hearts were exposed to ischemia/reperfusion test according to Langendorff. Thereafter mitochondria were isolated and the mitochondrial respiration was measured. Additionally, the ATP synthase activity measurements on the same preparations were done. Animals of all groups subjected to I/R exhibited a decreased state 3 respiration with the least change noted in DM+RPC group associated with no significant changes in state 2 respiration. In RPC, DM and DM+RPC group, no significant changes in the activity of ATP synthase were observed after I/R injury. These results suggest that the endogenous protective mechanisms of RPC and DM do preserve the mitochondrial function in heart when they act in combination.

Stimulation of mitochondrial ATP synthase activity - a new diazoxide-mediated mechanism of cardioprotection.

Pharmacological preconditioning by diazoxide and a model of experimental streptozotocin-induced acute diabetes mellitus (STZ-DM) provided similar levels of cardioprotection assessed as limiting myocardial infarct size. The aim was to explore the possibility of existence of another in vitro mechanism, which could be contributory to cardioprotection mediated by diazoxide treatment. Mitochondrial membrane fluidity and ATP synthase activity in isolated heart mitochondria were determined under the influence of two factors, STZ-DM condition and treatment with diazoxide. Both factors independently increased the ATP synthase activity (p<0.05), as no interaction effect was observed upon the combination of STZ-DM with diazoxide. On the other hand, the mitochondrial membrane fluidity was significantly increased by STZ-DM only; no significant main effect for diazoxide was found. Based on the results from measurements of enzyme kinetics, we assume a direct interaction of diazoxide with the molecule of ATP synthase stimulated its activity by noncompetitive activation. Our present work revealed, for the first time, that cardioprotection induced by diazoxide may not be caused exclusively by mitochondrial K(ATP) opening, but presumably also by a direct interaction of diazoxide with ATP synthase, although the mechanisms for achieving this activation cannot be fully delineated.

Remote preconditioning as a novel "conditioning" approach to repair the broken heart: potential mechanisms and clinical applications.

Remote ischemic preconditioning (RIPC) is a novel strategy of protection against ischemia-reperfusion (IR) injury in the heart (and/or other organs) by brief episodes of non-lethal IR in a distant organ/tissue. Importantly, RIPC can be induced noninvasively by limitation of blood flow in the extremity implying the applicability of this method in clinical situations. RIPC (and its delayed phase) is a form of relatively short-term adaptation to ischemia, similar to ischemic PC, and likely they both share triggering mechanisms, whereas mediators and end-effectors may differ. It is hypothesized that communication between the signals triggered in the remote organs and protection in the target organ may be mediated through substances released from the preconditioned organ and transported via the circulation (humoral pathways), by neural pathways and/or via systemic anti-inflammatory and antiapoptotic response to short ischemic bouts. Identification of molecules involved in RIPC cascades may have therapeutic and diagnostic implications in the management of myocardial ischemia. Elucidation of the mechanisms of endogenous cardioprotection triggered in the remote organ could lead to the development of diverse pharmacological RIPC mimetics. In the present article, the authors provide a short overview of RIPC-induced protection, proposed underlying mechanisms and factors modulating RIPC as a promising cardioprotective strategy.

Participation of heart mitochondria in myocardial protection against ischemia/reperfusion injury: benefit effects of short-term adaptation processes.

Acute streptozotocin diabetes mellitus (DM) as well as remote ischemic preconditioning (RPC) has shown a favorable effect on the postischemic-reperfusion function of the myocardium. Cardioprotective mechanisms offered by these experimental models involve the mitochondria with the changes in functional properties of membrane as the end-effector. The aim was to find out whether separate effects of RPC and DM would stimulate the mechanisms of cardioprotection to a maximal level or whether RPC and DM conditions would cooperate in stimulation of cardioprotection. Experiments were performed on male Wistar rats divided into groups: control, DM, RPC and DM treated by RPC (RPC+DM). RPC protocol of 3 cycles of 5-min hind limb ischemia followed by 5-min reperfusion was used. Ischemic-reperfusion injury was induced by 30-min ischemia followed by 40-min reperfusion of the hearts in Langendorff mode. Mitochondria were isolated by differential centrifugation, infarct size assessed by staining with 1 % 2,3,5-triphenyltetrazolium chloride, mitochondrial membrane fluidity with a fluorescent probe DPH, CoQ(9) and CoQ(10) with HPLC. Results revealed that RPC as well as DM decreased the infarct size and preserved mitochondrial function by increasing the mitochondrial membrane fluidity. Both used models separately offered a sufficient protection against ischemic-reperfusion injury without an additive effect of their combination.

Remote ischemic preconditioning of the heart: protective responses in functional and biophysical properties of cardiac mitochondria.

UNLABELLED: Remote ischemic preconditioning (RIP)-induced protection of myocardial energetics was well documented on the level of tissue, but data concerning the involvement of mitochondria were missing. We aimed at the identification of changes in membrane properties and respiratory functions induced in rat heart mitochondria by RIP. Experiments were performed on 46 male Wistar rats divided into control and RIP-treated groups of 21 animals each. Blood flow in the occluded area was recorded by MRI angiography in four animals. RIP protocol comprised of three successive 5-min occlusions each followed by 5-min reperfusions of descending branches of the right hind limb femoral artery. The efficacy of RIP was evaluated as the extent of RIP-induced protection against damage to the functions of mitochondria isolated by differential centrifugation after 30-min global ischemia followed by 40-min reperfusion of the hearts in Langendorff mode. ASSESSMENTS: mitochondrial membrane fluidity with a fluorescent probe DPH, CoQ(9) and CoQ(10) with HPLC, mitochondrial respiration with the Oxygraph-2k (Oroboros). Results revealed that RIP was affecting the mitochondria. The immediate protection conferred by RIP involves beneficial and prognostically significant effects: a total elimination of ischemia/reperfusion-induced depression of mitochondrial membrane fluidity and a trend for better preservation of mitochondrial state 3 respiration.

Treatment of rats with hypolipidemic compound pirinixic acid protects their hearts against ischemic injury: are mitochondrial K(ATP) channels and reactive oxygen species involved?

Hypolipidemic compound pirinixic acid (WY-14643, WY) is known to exert pleiotropic (other than primary) effects, such as activation of peroxisome proliferator-activated receptors (PPAR-alpha), transcription factors regulating different cardiac functions. Their role in ischemia-reperfusion (I/R) injury and cardioprotection is less clear, although protective effects of PPAR agonists have been documented. This study was designed to explore the effects of WY on the I/R injury in the rat heart and potential mechanisms involved, including mitochondrial K(ATP) channels (mitoK(ATP)) opening and production of reactive oxygen species (ROS). Langendorff-perfused hearts of rats intragastrally treated with WY (3 mg/kg/day) for 5 days and of control animals were subjected to 30-min global ischemia and 2-h reperfusion with or without 15-min perfusion with mitoK(ATP) blocker 5-hydroxydecanoate (5-HD) prior to I/R. Evaluation of the infarct size (IS, TTC staining) served as the main end-point of protection. Lipid peroxidation (a marker of ROS production) was determined by measurement of myocardial concentration of conjugated dienes (CD), whereas protein expression of endothelial NO synthase was analysed by Western blotting. A 2-fold increase in the cardiac protein levels of eNOS after treatment with WY was accompanied by lower post-I/R levels of CD compared with those in the hearts of untreated controls, although WY itself enhanced ROS generation prior to ischemia. IS was reduced by 47 % in the hearts of WY-treated rats (P<0.05), and this effect was reversed by 5-HD. Results suggest that PPAR-alpha activation may confer protection against lethal I/R injury in the rat heart that involves up-regulation of eNOS, mitoK(ATP) opening and reduced oxidative stress during I/R.