Jasna Marinovic

Age-related attenuation of isoflurane preconditioning in human atrial cardiomyocytes: roles for mitochondrial respiration and sarcolemmal adenosine triphosphate-sensitive potassium channel activity.

Background:Clinical trials suggest that anesthetic-induced preconditioning (APC) produces cardioprotection in humans, but the mechanisms of APC and significance of aging for APC in humans are not well understood. Here, the impact of age on the role of two major effectors of APC, mitochondria and sarcolemmal adenosine triphosphate–sensitive potassium (sarcKATP) channels, in preconditioning of the human atrial myocardium were investigated. Methods:Right atrial appendages were obtained from adult patients undergoing cardiac surgery and assigned to mid-aged (MA) and old-aged (OA) groups. APC was induced by isoflurane in isolated myocardium and isolated cardiomyocytes. Mitochondrial oxygen consumption measurements, myocyte survival testing, and patch clamp techniques were used to investigate mitochondrial respiratory function and sarcKATP channel activity. Results:After in vitro APC with isoflurane, the respiratory function of isolated mitochondria was better preserved after hypoxia–reoxygenation stress in MA than in OA. In isolated intact myocytes, APC significantly decreased oxidative stress–induced cell death in MA but not in OA, and isoflurane protection from cell death was attenuated by the sarcKATP channel inhibitor HMR-1098. Further, the properties of single sarcKATP channels were similar in MA and OA, and isoflurane sensitivity of pinacidil-activated whole cell KATP current was no different between MA and OA myocytes. Conclusion:Anesthetic-induced preconditioning with isoflurane decreases stress-induced cell death and preserves mitochondrial respiratory function to a greater degree in MA than in OA myocytes; however, sarcKATP channel activity is not differentially affected by isoflurane. Therefore, effectiveness of APC in humans may decrease with advancing age partly because of altered mitochondrial function of myocardial cells.

Volatile anesthetic-induced cardiac preconditioning

Pharmacological preconditioning with volatile anesthetics, or anesthetic-induced preconditioning (APC), is a phenomenon whereby a brief exposure to volatile anesthetic agents protects the heart from the potentially fatal consequences of a subsequent prolonged period of myocardial ischemia and reperfusion. Although not completely elucidated, the cellular and molecular mechanisms of APC appear to mimic those of ischemic preconditioning, the most powerful endogenous cardioprotective mechanism. This article reviews recently accumulated evidence underscoring the importance of mitochondria, reactive oxygen species, and KATP channels in cardioprotective signaling by volatile anesthetics. Moreover, the article addresses current concepts and controversies regarding the specific roles of the mitochondrial and the sarcolemmal KATP channels in APC.

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.

Distinct Roles for Sarcolemmal and Mitochondrial Adenosine Triphosphate-sensitive Potassium Channels in Isoflurane-induced Protection against Oxidative Stress

Background:Cardiac preconditioning, including that induced by halogenated anesthetics, is an innate protective mechanism against ischemia-reperfusion injury. The adenosine triphosphate-sensitive potassium (KATP) channels are considered essential in preconditioning mechanism. However, it is unclear whether KATP channels are triggers initiating the preconditioning signaling, and/or effectors responsible for the cardioprotective memory and activated during ischemia-reperfusion. Methods:Adult rat cardiomyocytes were exposed to oxidative stress with 200 &mgr;m H2O2 and 100 &mgr;m FeSO4. Myocyte survival was determined based on morphologic characteristics and trypan blue exclusion. To induce preconditioning, the myocytes were pretreated with isoflurane. The involvement of sarcolemmal and mitochondrial KATP channels was investigated using specific inhibitors HMR-1098 and 5-hydroxydecanoic acid. Data are expressed as mean ± SD. Results:Oxidative stress induced cell death in 47 ± 14% of myocytes. Pretreatment with isoflurane attenuated this effect to 26 ± 8%. Blockade of the sarcolemmal KATP channels abolished the protection by isoflurane pretreatment when HMR-1098 was applied throughout the experiment (50 ± 21%) or only during oxidative stress (50 ± 12%), but not when applied during isoflurane pretreatment (29 ± 13%). Inhibition of the mitochondrial KATP channels abolished cardioprotection irrespective of the timing of 5-hydroxydecanoic acid application. Cell death was 42 ± 23, 45 ± 23, and 46 ± 22% when 5-hydroxydecanoic acid was applied throughout the experiment, only during isoflurane pretreatment, or only during oxidative stress, respectively. Conclusion:The authors conclude that both sarcolemmal and mitochondrial KATP channels play essential and distinct roles in protection afforded by isoflurane. Sarcolemmal KATP channel seems to act as an effector of preconditioning, whereas mitochondrial KATP channel plays a dual role as a trigger and an effector.

Preconditioning by Isoflurane Induces Lasting Sensitization of the Cardiac Sarcolemmal Adenosine Triphosphate–sensitive Potassium Channel by a Protein Kinase C-δ–mediated Mechanism

Background:Cardioprotective effects of volatile anesthetics in anesthetic-induced preconditioning involve activation of the cardiac sarcolemmal adenosine triphosphate–sensitive potassium (sarcKATP) channels. This study addressed the memory phase of anesthetic preconditioning by investigating whether brief exposure to isoflurane produces lasting sensitization of the sarcKATP channel and whether protein kinase C mediates this effect. Methods:Whole cell sarcKATP channel current (IKATP) was monitored from single isolated rat ventricular cardiomyocytes. Pinacidil was used to open the channel, and the magnitude of activated IKATP was an indicator of channel’s ability to open. Involvement of protein kinase C was investigated using chelerythrine and isoform-specific peptide inhibitors and activators of protein kinase C-&dgr; and protein kinase C-ϵ. Results:The mean density of IKATP elicited by pinacidil (5 &mgr;m) in anesthetic-free conditions was 3.8 ± 3.7 pA/pF (n = 11). After 10 min of exposure to isoflurane (0.56 mm) and 10 or 30 min of anesthetic washout, pinacidil-elicited IKATP was increased to 15.6 ± 11.3 pA/pF (n = 12; P < 0.05) and 11.8 ± 3.9 pA/pF (n = 6; P < 0.05), respectively. In the presence of chelerythrine (5 &mgr;m), isoflurane did not potentiate channel opening, and IKATP was 6.6 ± 4.6 pA/pF (n = 11). Application of protein kinase C-&dgr; peptide inhibitor also abolished isoflurane-induced sensitization of sarcKATP channel, and IKATP was 7.7 ± 5.4 pA/pF (n = 12). In contrast, protein kinase C-ϵ peptide inhibitor did not affect channel sensitization, and pinacidil-elicited current was 14.8 ± 9.6 pA/pF (n = 12). Interestingly, when both protein kinase C-&dgr; and protein kinase C-ϵ activators were applied instead of isoflurane, they sensitized the channel to the same extent as isoflurane (18.9 ± 7.2 pA/pF, n = 11, and 18.6 ± 11.1 pA/pF, n = 10, respectively). Conclusion:Isoflurane induces prolonged sensitization of the sarcKATP channel to opening that persists even after anesthetic withdrawal. Our results indicate that protein kinase C-&dgr;, rather than protein kinase C-ϵ, is a likely mediator of isoflurane effects, although both protein kinase C-&dgr; and protein kinase C-ϵ can modulate the channel function.

Successive deep dives impair endothelial function and enhance oxidative stress in man

The aim of this study was to assess the effects of successive deep dives on endothelial function of large conduit arteries and plasma pro‐oxidant and antioxidant activity. Seven experienced divers performed six dives in six consecutive days using a compressed mixture of oxygen, helium and nitrogen (trimix) with diving depths ranging from 55 to 80 m. Before and after first, third and sixth dive, venous gas emboli formation and brachial artery function (flow‐mediated dilation, FMD) was assessed by ultrasound. In addition, plasma antioxidant capacity (AOC) was measured by ferric reducing antioxidant power, and the level of oxidative stress was assessed by thiobarbituric acid‐reactive substances (TBARS) method. Although the FMD was reduced to a similar extent after each dive, the comparison of predive FMD showed a reduction from 8·6% recorded before the first dive to 6·3% before the third (P = 0·03) and 5·7% before the sixth dive (P = 0·003). A gradual shift in baseline was also detected with TBARS assay, with malondialdehyde values increasing from 0·10 ± 0·02 μmol l−1 before the first dive to 0·16 ± 0·03 before the sixth (P = 0·005). Predive plasma AOC values also showed a decreasing trend from 0·67 ± 0·20 mmol l−1 trolox equivalents (first day) to 0·56 ± 0·12 (sixth day), although statistical significance was not reached (P = 0·08). This is the first documentation of acute endothelial dysfunction in the large conduit arteries occurring after successive deep trimix dives. Both endothelial function and plasma pro‐oxidant and antioxidant activity did not return to baseline during the course of repetitive dives, indicating possible cumulative and longer lasting detrimental effects.

Involuntary breathing movements improve cerebral oxygenation during apnea struggle phase in elite divers

We investigated whether the involuntary breathing movements (IBM) during the struggle phase of breath holding, together with peripheral vasoconstriction and progressive hypercapnia, have a positive effect in maintaining cerebral blood volume. The central hemodynamics, arterial oxygen saturation, brain regional oxyhemoglobin (bHbO(2)), deoxyhemoglobin, and total hemoglobin changes and IBM were monitored during maximal dry breath holds in eight elite divers. The frequency of IBM increased (by approximately 100%), and their duration decreased ( approximately 30%), toward the end of the struggle phase, whereas the amplitude was unchanged (compared with the beginning of the struggle phase). In all subjects, a consistent increase in brain regional deoxyhemoglobin and total hemoglobin was also found during struggle phase, whereas bHbO(2) changed biphasically: it initially increased until the middle of the struggle phase, with the subsequent relative decline at the end of the breath hold. Mean arterial pressure was elevated during the struggle phase, although there was no further rise in the peripheral resistance, suggesting unchanged peripheral vasoconstriction and implying the beneficial influence of the IBM on the cardiac output recovery (primarily by restoration of the stroke volume). The IBM-induced short-lasting, sudden increases in mean arterial pressure were followed by similar oscillations in bHbO(2). These results suggest that an increase in the cerebral blood volume observed during the struggle phase of dry apnea is most likely caused by the IBM at the time of the hypercapnia-induced cerebral vasodilatation and peripheral vasoconstriction.

High incidence of venous and arterial gas emboli at rest after trimix diving without protocol violations

SCUBA diving is associated with generation of gas emboli due to gas release from the supersaturated tissues during decompression. Gas emboli arise mostly on the venous side of circulation, and they are usually eliminated as they pass through the lung vessels. Arterialization of venous gas emboli (VGE) is seldom reported, and it is potentially related to neurological damage and development of decompression sickness. The goal of the present study was to evaluate the generation of VGE in a group of divers using a mixture of compressed oxygen, helium, and nitrogen (trimix) and to probe for their potential appearance in arterial circulation. Seven experienced male divers performed three dives in consecutive days according to trimix diving and decompression protocols generated by V-planner, a software program based on the Varying Permeability Model. The occurrence of VGE was monitored ultrasonographically for up to 90 min after surfacing, and the images were graded on a scale from 0 to 5. The performed diving activities resulted in a substantial amount of VGE detected in the right cardiac chambers and their frequent passage to the arterial side, in 9 of 21 total dives (42%) and in 5 of 7 divers (71%). Concomitant measurement of mean pulmonary artery pressure revealed a nearly twofold augmentation, from 13.6 ± 2.8, 19.2 ± 9.2, and 14.7 ± 3.3 mmHg assessed before the first, second, and the third dive, respectively, to 26.1 ± 5.4, 27.5 ± 7.3, and 27.4 ± 5.9 mmHg detected after surfacing. No acute decompression-related disorders were identified. The observed high gas bubble loads and repeated microemboli in systemic circulation raise questions about the possibility of long-term adverse effects and warrant further investigation.

Targeted expression of Kir6.2 in mitochondria confers protection against hypoxic stress

Selective K+ transport in the inner mitochondrial membrane has been attributed to at least three different channel types: ATP‐sensitive, Ca2+‐regulated and voltage‐dependent K+ channels. Studies utilizing their selective modulators have suggested that an increased activity of these channels plays an important role in the cellular protection from metabolic stress. However, direct evidence for this effect is largely absent, and recent findings on the lack of specificity for several channel openers and blockers have questioned the actual contribution of the mitochondrial K+ channels in the preservation of cellular viability. In order to directly investigate the role of enhanced mitochondrial K+ uptake in cellular protection, we selectively expressed the inward rectifying K+ channel Kir6.2 in the mitochondria of HEK293 and HL‐1 cells. Targeted Kir6.2 expression was achieved by cloning the Kir6.2 gene in pCMV/mito/GFP vector and the proper trafficking to mitochondria was confirmed by colocalization studies and Western blot. An increased K+ influx to mitochondria overexpressing Kir6.2, as evidenced by using the K+‐sensitive PBFI AM fluorescent dye, substantially improved the cellular viability after hypoxic stress, which was assessed by lactate dehydrogenase (LDH) release. In parallel, monitoring of mitochondrial Ca2+ during stress, via the specific indicator rhod‐2, revealed a significant attenuation of Ca2+ accumulation in mitochondria overexpressing K+ channels. This effect was abolished in mitochondria expressing an inactive mutant of Kir6.2. Mitochondria expressing Kir6.2 K+ channel also exhibited a significant degree of depolarization that became even more pronounced during the stress. In conclusion, this study provides the first non‐pharmacological evidence that an increased K+ influx to mitochondria protects against hypoxic stress by preventing detrimental effects of Ca2+ overload.

Role of sarcolemmal ATP-sensitive potassium channel in oxidative stress-induced apoptosis: mitochondrial connection

From time of their discovery, sarcolemmal ATP-sensitive K+ (sarcK ATP) channels were thought to have an important protective role in the heart during stress whereby channel opening protects the heart from stress-induced Ca2+ overload and resulting damage. In contrast, some recent studies indicate that sarcK ATP channel closing can lead to cardiac protection. Also, the role of the sarcK ATP channel in apoptotic cell death is unclear. In the present study, the effects of channel inhibition on apoptosis and the specific interaction between the sarcK ATP channel and mitochondria were investigated. Apoptotic cell death of cultured HL-1 and neonatal cardiomyocytes following exposure to oxidative stress was significantly increased in the presence of sarcK ATP channel inhibitor HMR-1098 as evidenced by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and caspase-3,7 assays. This was paralleled by an increased release of cytochrome c from mitochondria to cytosol, suggesting activation of the mitochondrial death pathway. sarcK ATP channel inhibition during stress had no effect on Bcl-2, Bad, and phospho-Bad, indicating that the increase in apoptosis cannot be attributed to these modulators of the apoptotic pathway. However, monitoring of mitochondrial Ca2+ with rhod-2 fluorescent indicator revealed that mitochondrial Ca2+ accumulation during stress is potentiated in the presence of HMR-1098. In conclusion, this study provides novel evidence that opening of sarcK ATP channels, through a specific Ca2+-related interaction with mitochondria, plays an important role in preventing cardiomyocyte apoptosis and mitochondrial damage during stress.