Danijel Pravdic

Anesthetic-induced preconditioning delays opening of mitochondrial permeability transition pore via protein kinase C-ε mediated pathway

Background:Cardioprotection by volatile anesthetic-induced preconditioning (APC) involves activation of protein kinase C (PKC). This study investigated the importance of APC-activated PKC in delaying mitochondrial permeability transition pore (mPTP) opening. Methods:Rat ventricular myocytes were exposed to isoflurane in the presence or absence of nonselective PKC inhibitor chelerythrine or isoform-specific inhibitors of PKC-&dgr; (rottlerin) and PKC-ϵ (myristoylated PKC-ϵ V1–2 peptide), and the mPTP opening time was measured by using confocal microscopy. Ca2+-induced mPTP opening was measured in mitochondria isolated from rats exposed to isoflurane in the presence and absence of chelerythrine or in mitochondria directly treated with isoflurane after isolation. Translocation of PKC-ϵ was assessed in APC and control cardiomyocytes by Western blotting. Results:In cardiomyocytes, APC prolonged time necessary to induce mPTP opening (261 ± 26 s APC vs. 216 ± 27 s control; P < 0.05), and chelerythrine abolished this delay to 213 ± 22 s. The effect of isoflurane was also abolished when PKC-ϵ inhibitor was applied (210 ± 22 s) but not in the presence of PKC-&dgr; inhibitor (269 ± 31 s). Western blotting revealed translocation of PKC-ϵ toward mitochondria in APC cells. The Ca2+ concentration required for mPTP opening was significantly higher in mitochondria from APC rats (45 ± 8 &mgr;m · mg−1 control vs. 64 ± 8 &mgr;m · mg−1 APC), and APC effect was reversed with chelerythrine. In contrast, isoflurane did not protect directly treated mitochondria. Conclusion:APC induces delay of mPTP opening through PKC-ϵ–mediated inhibition of mPTP opening, but not through PKC-&dgr;. These results point to the connection between cytosolic and mitochondrial components of cardioprotection by isoflurane.

Isoflurane postconditioning protects against reperfusion injury by preventing mitochondrial permeability transition by an endothelial nitric oxide synthase-dependent mechanism.

Background:The role of endothelial nitric oxide synthase (eNOS) in isoflurane postconditioning (IsoPC)-elicited cardioprotection is poorly understood. The authors addressed this issue using eNOS−/− mice. Methods:In vivo or Langendorff-perfused mouse hearts underwent 30 min of ischemia followed by 2 h of reperfusion in the presence and absence of postconditioning produced with isoflurane 5 min before and 3 min after reperfusion. Ca2+-induced mitochondrial permeability transition (MPT) pore opening was assessed in isolated mitochondria. Echocardiography was used to evaluate ventricular function. Results:Postconditioning with 0.5, 1.0, and 1.5 minimum alveolar concentrations of isoflurane decreased infarct size from 56 ± 10% (n = 10) in control to 48 ± 10%, 41 ± 8% (n = 8, P < 0.05), and 38 ± 10% (n = 8, P < 0.05), respectively, and improved cardiac function in wild-type mice. Improvement in cardiac function by IsoPC was blocked by NG-nitro-l-arginine methyl ester (a nonselective nitric oxide synthase inhibitor) administered either before ischemia or at the onset of reperfusion. Mitochondria isolated from postconditioned hearts required significantly higher in vitro Ca2+ loading than did controls (78 ± 29 &mgr;m vs. 40 ± 25 &mgr;m CaCl2 per milligram of protein, n = 10, P < 0.05) to open the MPT pore. Hearts from eNOS−/− mice displayed no marked differences in infarct size, cardiac function, and sensitivity of MPT pore to Ca2+, compared with wild-type hearts. However, IsoPC failed to alter infarct size, cardiac function, or the amount of Ca2+ necessary to open the MPT pore in mitochondria isolated from the eNOS−/− hearts compared with control hearts. Conclusions:IsoPC protects mouse hearts from reperfusion injury by preventing MPT pore opening in an eNOS-dependent manner. Nitric oxide functions as both a trigger and a mediator of cardioprotection produced by IsoPC.

Mitochondrial depolarization underlies delay in permeability transition by preconditioning with isoflurane: roles of ROS and Ca2+

During reperfusion, the interplay between excess reactive oxygen species (ROS) production, mitochondrial Ca(2+) overload, and mitochondrial permeability transition pore (mPTP) opening, as the crucial mechanism of cardiomyocyte injury, remains intriguing. Here, we investigated whether an induction of a partial decrease in mitochondrial membrane potential (DeltaPsi(m)) is an underlying mechanism of protection by anesthetic-induced preconditioning (APC) with isoflurane, specifically addressing the interplay between ROS, Ca(2+), and mPTP opening. The magnitude of APC-induced decrease in DeltaPsi(m) was mimicked with the protonophore 2,4-dinitrophenol (DNP), and the addition of pyruvate was used to reverse APC- and DNP-induced decrease in DeltaPsi(m). In cardiomyocytes, DeltaPsi(m), ROS, mPTP opening, and cytosolic and mitochondrial Ca(2+) were measured using confocal microscope, and cardiomyocyte survival was assessed by Trypan blue exclusion. In isolated cardiac mitochondria, antimycin A-induced ROS production and Ca(2+) uptake were determined spectrofluorometrically. In cells exposed to oxidative stress, APC and DNP increased cell survival, delayed mPTP opening, and attenuated ROS production, which was reversed by mitochondrial repolarization with pyruvate. In isolated mitochondria, depolarization by APC and DNP attenuated ROS production, but not Ca(2+) uptake. However, in stressed cardiomyocytes, a similar decrease in DeltaPsi(m) attenuated both cytosolic and mitochondrial Ca(2+) accumulation. In conclusion, a partial decrease in DeltaPsi(m) underlies cardioprotective effects of APC by attenuating excess ROS production, resulting in a delay in mPTP opening and an increase in cell survival. Such decrease in DeltaPsi(m) primarily attenuates mitochondrial ROS production, with consequential decrease in mitochondrial Ca(2+) uptake.

Isoflurane differentially modulates mitochondrial reactive oxygen species production via forward versus reverse electron transport flow: Implications for preconditioning

Background: Reactive oxygen species (ROS) mediate the effects of anesthetic precondition to protect against ischemia and reperfusion injury, but the mechanisms of ROS generation remain unclear. In this study, the authors investigated if mitochondria-targeted antioxidant (mitotempol) abolishes the cardioprotective effects of anesthetic preconditioning. Further, the authors investigated the mechanism by which isoflurane alters ROS generation in isolated mitochondria and submitochondrial particles. Methods: Rats were pretreated with 0.9% saline, 3.0 mg/kg mitotempol in the absence or presence of 30 min exposure to isoflurane. Myocardial infarction was induced by left anterior descending artery occlusion for 30 min followed by reperfusion for 2 h and infarct size measurements. Mitochondrial ROS production was determined spectrofluorometrically. The effect of isoflurane on enzymatic activity of mitochondrial respiratory complexes was also determined. Results: Isoflurane reduced myocardial infarct size (40 ± 9% = mean ± SD) compared with control experiments (60 ± 4%). Mitotempol abolished the cardioprotective effects of anesthetic preconditioning (60 ± 9%). Isoflurane enhanced ROS generation in submitochondrial particles with nicotinamide adenine dinucleotide (reduced form), but not with succinate, as substrate. In intact mitochondria, isoflurane enhanced ROS production in the presence of rotenone, antimycin A, or ubiquinone when pyruvate and malate were substrates, but isoflurane attenuated ROS production when succinate was substrate. Mitochondrial respiratory experiments and electron transport chain complex assays revealed that isoflurane inhibited only complex I activity. Conclusions: The results demonstrated that isoflurane produces ROS at complex I and III of the respiratory chain via the attenuation of complex I activity. The action on complex I decreases unfavorable reverse electron flow and ROS release in myocardium during reperfusion.

Differences in Production of Reactive Oxygen Species and Mitochondrial Uncoupling as Events in the Preconditioning Signaling Cascade Between Desflurane and Sevoflurane

BACKGROUND: Signal transduction cascade of anesthetic-induced preconditioning has been extensively studied, yet many aspects of it remain unsolved. Here, we investigated the roles of reactive oxygen species (ROS) and mitochondrial uncoupling in cardiomyocyte preconditioning by two modern volatile anesthetics: desflurane and sevoflurane. METHODS: Adult rat ventricular cardiomyocytes were isolated enzymatically. The preconditioning potency of desflurane and sevoflurane was assessed in cell survival experiments by evaluating myocyte protection from the oxidative stress-induced cell death. ROS production and flavoprotein fluorescence, an indicator of flavoprotein oxidation and mitochondrial uncoupling, were monitored in real time by confocal microscopy. The functional aspect of enhanced ROS generation by the anesthetics was assessed in cell survival and confocal experiments using the ROS scavenger Trolox. RESULTS: Preconditioning of cardiomyocytes with desflurane or sevoflurane significantly decreased oxidative stress-induced cell death. That effect coincided with increased ROS production and increased flavoprotein oxidation detected during acute myocyte exposure to the anesthetics. Desflurane induced significantly greater ROS production and flavoprotein oxidation than sevoflurane. ROS scavenging with Trolox abrogated preconditioning potency of anesthetics and attenuated flavoprotein oxidation. CONCLUSION: Preconditioning with desflurane or sevoflurane protects isolated rat cardiomyocytes from oxidative stress-induced cell death. Scavenging of ROS abolishes the preconditioning effect of both anesthetics and attenuates anesthetic-induced mitochondrial uncoupling, suggesting a crucial role for ROS in anesthetic-induced preconditioning and implying that ROS act upstream of mitochondrial uncoupling. Desflurane exhibits greater effect on stimulation of ROS production and mitochondrial uncoupling than sevoflurane.

Suppressed Ca2+/CaM/CaMKII-dependent KATP channel activity in primary afferent neurons mediates hyperalgesia after axotomy

Painful axotomy decreases KATP channel current (IKATP) in primary afferent neurons. Because cytosolic Ca2+ signaling is depressed in injured dorsal root ganglia (DRG) neurons, we investigated whether Ca2+–calmodulin (CaM)–Ca2+/CaM-dependent kinase II (CaMKII) regulates IKATP in large DRG neurons. Immunohistochemistry identified the presence of KATP channel subunits SUR1, SUR2, and Kir6.2 but not Kir6.1, and pCaMKII in neurofilament 200–positive DRG somata. Single-channel recordings from cell-attached patches revealed that basal and evoked IKATP by ionomycin, a Ca2+ ionophore, is activated by CaMKII. In axotomized neurons from rats made hyperalgesic by spinal nerve ligation (SNL), basal KATP channel activity was decreased, and sensitivity to ionomycin was abolished. Basal and Ca2+-evoked KATP channel activity correlated inversely with the degree of hyperalgesia induced by SNL in the rats from which the neurons were isolated. Inhibition of IKATP by glybenclamide, a selective KATP channel inhibitor, depolarized resting membrane potential (RMP) recorded in perforated whole-cell patches and enhanced neurotransmitter release measured by amperometry. The selective KATP channel opener diazoxide hyperpolarized the RMP and attenuated neurotransmitter release. Axotomized neurons from rats made hyperalgesic by SNL lost sensitivity to the myristoylated form of autocamtide-2-related inhibitory peptide (AIPm), a pseudosubstrate blocker of CaMKII, whereas axotomized neurons from SNL animals that failed to develop hyperalgesia showed normal IKATP inhibition by AIPm. AIPm also depolarized RMP in control neurons via KATP channel inhibition. Unitary current conductance and sensitivity of KATP channels to cytosolic ATP and ligands were preserved even after painful nerve injury, thus providing opportunities for selective therapeutic targeting against neuropathic pain.

Isoflurane protects cardiomyocytes and mitochondria by immediate and cytosol‐independent action at reperfusion

Background and purpose:  The volatile anaesthetic isoflurane protects the heart from ischaemia and reperfusion (I/R) injury when applied at the onset of reperfusion [anaesthetic postconditioning (APoC)]. However, the mechanism of APoC‐mediated protection is unknown. In this study, we examined the effect of APoC on mitochondrial bioenergetics, mitochondrial matrix pH (pHm) and cytosolic pH (pHi), and intracellular Ca2+.

Survival of neurally induced mesenchymal cells may determine degree of motor recovery in injured spinal cord rats

PURPOSE We recently developed a new method for efficient generation of neural-like cells from mice bone marrow (BM)-derived mesenchymal stem cells (MSC) by exposing MSCs to epigenetic modifiers and a neural stem cell environment. These neurally induced MSCs (NI-MSCs) differentiate into neuronal- and glial-like cells in vitro, release neurotrophic factors NGF and BDNF, survive and integrate after transplantation in intact spinal cord. The aim of this study was to determine whether transplanted NI-MSCs survive, differentiate, and integrate in injured spinal cord (ISC) rats and promote functional recovery. METHODS Twenty rats, half grafted with MSCs and half with NI-MSCs, were used for survival and differentiation studies. Results were analyzed using triple-labeled immunohistochemistry. For motor function studies the 3 group of adult female Sprague Dawley rats received PBS (vehicle), MSCs, or NI-MSCs, respectively. Functional outcome was measured using the BBB scale. RESULTS Results demonstrated gradual improvement of locomotor function in NI-MSC-transplanted rats in comparison to vehicle and non-modified MSC-transplanted animals, with statistically significant differences at 7, 14, and 21 days post transplantation. Immunocytochemical studies revealed poor survival of NI-MSCs within the ISC as early as 3 weeks after transplantation. CONCLUSIONS Thus, there is a correlation between the degree of surviving NI-MSCs and extent of functional recovery.

Monitoring Mitochondrial Electron Fluxes Using NAD(P)H-Flavoprotein Fluorometry Reveals Complex Action of Isoflurane on Cardiomyocytes

Mitochondrial bioenergetic studies mostly rely on isolated mitochondria thus excluding the regulatory role of other cellular compartments important for the overall mitochondrial function. In intact cardiomyocytes, we followed the dynamics of electron fluxes along specific sites of the electron transport chain (ETC) by simultaneous detection of NAD(P)H and flavoprotein (FP) fluorescence intensities using a laser-scanning confocal microscope. This method was used to delineate the effects of isoflurane, a volatile anesthetic and cardioprotective agent, on the ETC. Comparison to the effects of well-characterized ETC inhibitors and uncoupling agent revealed two distinct effects of isoflurane: uncoupling-induced mitochondrial depolarization and inhibition of ETC at the level of complex I. In correlation, oxygen consumption measurements in cardiomyocytes confirmed a dose-dependent, dual effect of isoflurane, and in isolated mitochondria an obstruction of the ETC primarily at the level of complex I. These effects are likely responsible for the reported mild stimulation of mitochondrial reactive oxygen species (ROS) production required for the cardioprotective effects of isoflurane. In conclusion, isoflurane exhibits complex effects on the ETC in intact cardiomyocytes, altering its electron fluxes, and thereby enhancing ROS production. The NAD(P)H-FP fluorometry is a useful method for exploring the effect of drugs on mitochondria and identifying their specific sites of action within the ETC of intact cardiomyocytes.

Cardiac-specific overexpression of GTP cyclohydrolase 1 restores ischaemic preconditioning during hyperglycaemia

AIMS Hyperglycaemia (HG) decreases intracellular tetrahydrobiopterin (BH(4)) concentrations, and this action may contribute to injury during myocardial ischaemia and reperfusion. We investigated whether increased BH(4) by cardiomyocyte-specific overexpression of the GTP cyclohydrolase (GTPCH) 1 gene rescues myocardial and mitochondrial protection by ischaemic preconditioning (IPC) during HG through a nitric oxide (NO)-dependent pathway. METHODS AND RESULTS Mice underwent 30 min of myocardial ischaemia followed by 2 h of reperfusion with or without IPC elicited with four cycles of 5 min ischaemia/5 min of reperfusion in the presence or absence of HG produced by d-glucose. In C57BL/6 wild-type mice, IPC increased myocardial BH(4) and NO concentrations and decreased myocardial infarct size (30 ± 3% of risk area) compared with control (56 ± 5%) experiments. This protective effect was inhibited by HG (48 ± 3%) but not hyperosmolarity. GTPCH-1 overexpression increased myocardial BH(4) and NO concentrations and restored cardioprotection by IPC during HG (32 ± 4%). In contrast, a non-selective NO synthase inhibitor N(G)-nitro-l-arginine methyl ester attenuated the favourable effects of GTPCH-1 overexpression (52 ± 3%) during HG. Mitochondria isolated from myocardium subjected to IPC required significantly higher in vitro Ca(2+) concentrations (184 ± 14 µmol mg(-1) protein) to open the mitochondrial permeability transition pore when compared with mitochondria isolated from control experiments (142 ± 10 µmol mg(-1) protein). This beneficial effect of IPC was reversed by HG and rescued by GTPCH-1 overexpression. CONCLUSION Increased BH(4) by cardiomyocyte-specific overexpression of GTPCH-1 preserves the ability of IPC to elicit myocardial and mitochondrial protection that is impaired by HG, and this action appears to be dependent on NO.