mitoKATP Channel Research Articles

Enhancement of Liver Regeneration by Adenosine Triphosphate-Sensitive K+ Channel Opener (Diazoxide) After Partial Hepatectomy

Enhancement of liver regeneration is a matter of importance after partial liver transplantation including small-for-size grafting. Mitochondrial adenosine triphosphate (ATP)-sensitive K⁺ (mitoKATP) channel plays an important role in mitochondrial bioenergetics, which is a prerequisite for liver regeneration. However, the ATP-sensitive K⁺ (KATP) channel in hepatocytes is incompletely understood. We investigated the KATP channel in hepatocytes and examined the effects of diazoxide, a potent KATP channel opener, on liver regeneration using a rat model. Using rat primary hepatocytes, expression and localization of KATP channel subunits, Kir6.x and sulfonylurea receptor (SUR)x, were studied by polymerase chain reaction, Western blotting, and immunostaining. To investigate the role of KATP channel openers in liver regeneration, we allocated rats into four groups: control (vehicle) (n=24), diazoxide (n=24), vehicle plus channel blocker (n=6), and diazoxide plus channel blocker (n=6) groups. After 70% partial hepatectomy, hepatic tissue ATP levels, liver-to-body weight ratio, and proliferation rate of hepatocytes were examined. KATP channel subunits, Kir6.1 and SUR1, were detected on hepatic mitochondria. During liver regeneration, liver-to-body weight ratio, proliferation rate of hepatocytes, and the hepatic ATP level were significantly higher in the diazoxide group than the control group at 2 days after partial hepatectomy. These effects of diazoxide were neutralized by a KATP channel blocker. We demonstrated the existence of a mitoKATP channel in hepatocytes composed of Kir6.1 and SUR1. Diazoxide could enhance liver regeneration by keeping a higher ATP content of the liver tissue. These results suggest that diazoxide will sustain the mitochondrial energetics through the mitoKATP channel opening.

Ischemia reperfusion injury, KATPchannels, and exercise-induced cardioprotection against apoptosis

Exercise is a potent stimulus against cardiac ischemia reperfusion (IR) injury, although the protective mechanisms are not completely understood. The study purpose was to examine whether the mitochondrial or sarcolemmal ATP-sensitive potassium channel (mito K(ATP) or sarc K(ATP), respectively) mediates exercise-induced cardioprotection against post-IR cell death and apoptosis. Eighty-six, 4-mo-old male Sprague Dawley rats were randomly assigned to treadmill exercise (Ex; 30 m/min, 3 days, 60 min, ∼70 maximal oxygen uptake) and sedentary (Sed) treatments. Rats were exposed to regional cardiac ischemia (50 min) and reperfusion (120 min) or Sham (170 min; no ligation) surgeries. Exercise subgroups received placebo (saline), 5-hydroxydecanoate (5HD; 10 mg/kg ip), or HMR1098 (10 mg/kg ip) to inhibit mito K(ATP) or sarc K(ATP) channel. Comprehensive outcome assessments included post-IR ECG arrhythmias, cardiac tissue necrosis, redox perturbations, and autophagy biomarkers. No arrhythmia differences existed between exercised and sedentary hearts following extended-duration IR (P < 0.05). The sarc K(ATP) channel was confirmed essential (P = 0.002) for prevention of antinecrotic tissue death with exercise (percent infarct, Sed = 42%; Ex = 20%; Ex5HD = 16%; ExHMR = 42%), although neither the mito K(ATP) (P = 0.177) nor sarc K(ATP) (P = 0.274) channel provided post-IR protection against apoptosis (terminal deoxynucleotidyl transferase deoxy UTP-mediated nick-end labeling-positive nuclei/mm(2), Sham = 1.8 ± 0.5; Sed = 19.4 ± 6.7; Ex = 7.5 ± 4.6; Ex5HD = 14.0 ± 3.9; ExHMR = 11.1 ± 1.8). Exercise preconditioning also appears to preserve basal autophagy levels, as assessed by Beclin 1 (P ≤ 0.001), microtubule-associated protein-1 light-chain 3B ratios (P = 0.020), and P62 (P ≤ 0.001), in the hours immediately following IR. Further research is needed to better understand these findings and corresponding redox changes in exercised hearts.

Mitochondrial K+ channels are involved in ischemic postconditioning in rat hearts

The mitochondrial calcium-activated potassium channel (mitoK(Ca)) and the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) are both involved in cardiac preconditioning. Here, we examined whether these two channels are also involved in ischemic or pharmacological postconditioning. Using Langendorff perfusion, rat hearts were made hypoxic for 45 min and then reoxygenated for 30 min. Ischemic postconditioning (IPT) was achieved through application of 3 cycles of 10 s of reperfusion and 10 s of ischemia before reoxygenation, with and without paxilline (Pax; a mitoK(Ca) blocker) or 5-hydroxydecanoate (5-HD; a mitoK(ATP) blocker). Pharmacological postconditioning was carried out for 5 min at the onset of reoxygenation using NS1619 (a mitoK(Ca) opener) or diazoxide (Dia; a mitoK(ATP) opener). Pax and 5-HD abolished IPT-induced cardioprotection from reoxygenation injury, whereas administration of NS1619 or Dia significantly improved cardiac contractile activity and reduced aspartate aminotransferase (an index of myocyte injury) release following reoxygenation. In addition, isolated rat myocytes were loaded with tetramethylrhodamine methyl ester (TMRE; fluorescent mitochondrial membrane potential indicator) and 2',7'-dichlorofluorescein [DCFH; fluorescent reactive oxygen species (ROS) indicator] or Fluo-4-acetoxymethyl ester (Fluo-4-AM; fluorescent calcium indicator). When TMRE-loaded myocytes were laser illuminated, the DCFH and Fluo-4 fluorescence increased, and TMRE fluorescence decreased. These effects were significantly inhibited by NS1619 and Dia. We therefore conclude that IPT may protect the heart through activation of mitoK(ATP) and mitoK(Ca) channels, and that opening of these channels at the onset of reoxygenation protects the heart from reoxygenation injury, most likely by reducing excess generation of ROS and the resultant Ca(2+) overload.

Oxytocin protects cardiomyocytes from apoptosis induced by ischemia–reperfusion in rat heart: Role of mitochondrial ATP-dependent potassium channel and permeability transition pore

The current study examines the protective effect of oxytocin (OT) on cardiomyocyte apoptosis modulated by mitochondrial ATP-dependent potassium (mitoKATP) channel and permeability transition pore (mPTP) in the preconditioned myocardium of anesthetized rats. Eighty rats were equally divided into eight groups. The hearts of all animals except for the sham group were subjected to 25min ischemia and 120min reperfusion. Oxytocin, 5-hydroxydeconoate (5-HD), a specific inhibitor of the mitoKATP channel, and atractyloside (ATRC), an mPTP opener, were used prior to ischemia. Hemodynamic parameters were recorded throughout the experiment. Evaluations were made by infarct size, plasma lactate dehydrogenase level (LDH), transmission electron microscopy (TEM) and immunohistochemistry studies. OT prevented mean arterial pressure drop during early phase of ischemia and reperfusion. Treatment with OT before IR induction normalizes cardiomyocytes both in light microscopy and TEM observations. In addition, OT significantly reduced TUNEL- and increased Bcl-2-labeled positive cell number relative to IR (p<0.05). However, 5HD or ATRC inhibited the protective effects of OT on cardiomyocytes damaged by IR (p<0.05). Ultrastructural changes including extensive myofibril loss, sarcolemmal disruption and mitochondrial swelling due to amorphous dens bodies indicate necrosis induction in 5HD and ATRC as well as in IR groups. Restoration of immunohistochemistry parameters and protection against IR-induced ultrastructural changes confirm OT cardioprotective effects via mitoKATP channel and mPTP modulation in apoptosis induced by ischemia–reperfusion.

Differential expression of mitoKATP subunits in cardiac mitochondrial subpopulations and the influence of Type I diabetes

Mitochondrial dysfunction is an underlying risk for heart failure in Type I diabetes mellitus. MitoKATP channels are likely composed of an inwardly rectifying K+ channel subunit (Kir6.1) and a sulphonylurea receptor (SUR1). MitoKATP regulate mitochondrial volume and membrane potential: two functions disturbed by diabetes mellitus. In the heart, two mitochondrial populations exist: subsarcolemmal (SSM) and interfibrillar (IFM) which differ in their subcellular locations. It is not known whether: a) Kir6.1 or SUR1 expression is similar in IFM and SSM or whether b) diabetes mellitus alters the expression of Kir6.1 or SUR1 subunits in either SSM or IFM. Male FVB mice were made diabetic via multiple low‐dose injections of streptozoticin and examined after 5 weeks. Western blot analyses on cardiac mitochondrial subpopulations revealed Kir6.1 expression in non‐diabetic IFM was greater relative to SSM (p &lt; 0.05), but SUR1 expression was equivalent. Kir6.1 protein was reduced (p &lt; 0.05) in the IFM of diabetic mice. In contrast, diabetes mellitus had no effect on Kir6.1 protein expression in SSM or SUR1 expression in either IFM or SSM. These data show that expression Kir6.1 may be higher in IFM than SSM and that diabetes mellitus reduces Kir6.1 expression in IFM. This suggests that mitoKATP components may be differentially impacted by diabetes mellitus and that these effects may be specific to a subset of mitochondria.

REDUCTION OF FATIGUE IN SKELETAL MUSCLE BY DIAZOXIDE: POSSIBLE PARTICIPATION OF MITOKATP.

The decrease in the ATP has an important role in the development of muscle fatigue and any event related to alteration of production of energy, this determine the tendency of muscle fatigue (Allen et al. 2008). KATP channels have been described in the inner mitochondrial membrane (mitoKATP) (Inoue et al. 1991). Garcia et al. (2009) showed that mitoKATP plays an important role in protecting the muscle during fatigue. The intention of this study was to determine the possible participation of mitoKATP channels during fatigue exploring the effect of Diazoxide (a selective activator) and selective inhibitor 5‐HD, in tension of slow skeletal muscle of chicken in a in vitro model of fatigue.Anterior Latissimus Dorsi (ALD) muscle of Arbor acres variety chicken was used. The muscle was mounted on an experimental recording chamber by mecanoelectric transducer (Grass FT03), which through an amplifier and a analog‐digital interface (Digidata 1322A) allowed to acquire the muscle tension generated by a computer (Pentium 4) and a “software” of data acquisition (pCLAMP 9.2). The muscle was stimulated until the force decreased by 70‰ repetitive electrical stimulation, then applied Diazoxide for 6 min to observe its effect (10, 30 and 100 μM, and 500 μM of 5‐HD.In a Diazoxide dose‐response curve, effect of 30 μM increased postfatige tension (40.67±8‰), combination 5‐HD with Diazoxide had no significant effect in fatigue. The results shows a protective effect of muscle by Diazoxide, however the lack of effect of 5‐HD may be due an unspecific interaction with channels of skeletal muscle.

Sevoflurane-induced delayed neuroprotection involves mitoKATP channel opening and PKC ε activation

There is an increasing body of evidence that a brief exposure to anesthesia induces ischemic tolerance in rat brain (anesthetic preconditioning). However, it is unknown whether preconditioning with sevoflurane, a commonly used volatile anesthetic in current clinical practice, produces a delayed window of neuroprotection against ischemia and what the mechanisms are for this protection. To address these issues, adult male Sprague-Dawley rats were subjected to middle cerebral arterial occlusion (MCAO) for 2h. Sevoflurane preconditioning was induced 24h before brain ischemia by exposing the animals to sevoflurane at 1.0 minimum alveolar concentration (2.4%) in oxygen for 60min. Animals preconditioned with sevoflurane had lower neurological deficit scores and smaller brain infarct volumes than animals with brain ischemia at 6 and 24h after MCAO, respectively. Application of a selective antagonist for mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel, 5-hydroxydecanoate (5-HD, 40mg/kg i.p.) 30min before sevoflurane exposure attenuated this beneficial effect. Moreover, protein kinase C ε (PKC ε) was translocated to the membrane fraction at 6h, but not 24h, after brain reperfusion in animals preconditioned with sevoflurane and this effect was also abolished by 5-HD. We concluded that sevoflurane preconditioning induces a delayed neuroprotection and that mitochondrial K(ATP) channels and PKC ε may be involved in this neuroprotection.

Fibroblast growth factor-2-induced cardioprotection against myocardial infarction occurs via the interplay between nitric oxide, protein kinase signaling, and ATP-sensitive potassium channels

Fibroblast growth factor-2 (FGF2) protects the heart from ischemia–reperfusion (I-R) injury via a vast network of protein kinases. In the heart, downstream effectors of these FGF2-triggered signals have not yet been identified. It is hypothesized that nitric oxide (NO) signaling and ATP-sensitive potassium (KATP) channel activity are key effectors of protein kinases activated by FGF2-mediated cardioprotection. Hearts with a cardiac-specific overexpression of FGF2 (FGF2 Tg) were subjected to I-R injury in the absence or the presence of selective inhibitors of NO synthase (NOS) isoforms or sarcolemmal (sarcKATP) and mitochondrial (mitoKATP) KATP channels. Multiple NOS isoforms are necessary for FGF2-mediated cardioprotection, and nitrite levels are significantly reduced in FGF2 Tg hearts upon inhibition of protein kinase C or mitogen-activated protein kinases. Likewise, sarcKATP and mitoKATP channels are important for cardioprotection elicited by endogenous FGF2. These findings suggest that FGF2-induced cardioprotection occurs via protein kinase-NOS pathways as well as KATP channel activity.

Direct action of angiotensin II on the conduction through papillary muscle preparations of rat heart immediately after reoxygenation

We investigated the direct action of angiotensin II (Ang II) on myocardial conduction and transmembrane action potential immediately after reoxygenation. MethodAfter superfusion in a simulated ischemic solution, ventricular papillary muscle preparations of rat heart were washed with oxygenated Tyrode solution containing Ang II, Ang II plus CV-11974 (an AT1 receptor blocker), or Ang II plus 5-hydroxydecanoic acid (mito-KATP blocker), under rapid electrical stimulation (RES) for 60s. ResultsIn the control experiments, the incidence of conduction delay and block was the highest within the first 10s, and subsequently, 1:1 conduction was established after 40s. Ang II significantly enhanced the 2:1 conduction block during the later phase of RES (40–60s after reoxygenation). This effect of Ang II was abolished by either CV-11974 (P<0.001) or 5-HD (P<0.001). ConclusionRES-induced conduction delay and block immediately after reoxygenation were accelerated by Ang II, which could be relevant to the maintenance of reperfusion arrhythmias. The mito-KATP channel may participate in the mechanism underlying this phenomenon.

Diazoxide Pretreatment Enhances L6 Skeletal Myoblast Survival and Inhibits Apoptosis Induced by Hydrogen Peroxide

Skeletal myoblast (SKM) transplantation is a promising approach to regenerate tissue and improve the function of the injured heart. However, the number of survival cells transplanted to host myocardium is quite poor due to high rate of apoptosis; diazoxide (DZ) is a highly selective mito-KATP channel opener that may reduce cell apoptosis by relieving reactive oxygen species (ROS) damage. The aim of this study is to explore the protective effects of DZ on L6 SKM damage induced by hydrogen peroxide (H(2)O(2) ) in vitro. Different dose and time of H(2)O(2) and DZ treatment were performed and only 24 hr of 1.00 mmol/L H(2) O(2) treatment and 200 μmol/L DZ pretreatment for 30 min were used for further experiment. L6 SKMs were cultured and divided into control group (no treatment), H(2)O(2) group (24 hr of 1.00 mmol/L H(2) O(2) treatment) and DZ + H(2)O(2) group (pretreated with 200 μmol/L DZ for 30 min before 24 hr of 1.00 mmol/L H(2) O(2) treatment). Compared with control group, H(2)O(2) treatment caused cell damage, increased lactate dehydrogenase release, cell apoptosis, and bax gene expression, while reduced cell proliferation and decreased bcl-2 expression. DZ pretreatment may protect cells from damage induced by H(2)O(2) and reduce cell apoptosis by increasing bcl-2 and decreasing bax expression. DZ pretreatment may also promote cell proliferation measured by both PCNA expression and flow cytometry method. These results suggest that DZ may protect L6 SKMs from damage induced by H(2)O(2) by maintaining integrity of cell membrane, reducing apoptosis and increasing proliferation in vitro.

GSK3β Transfers Cytoprotective Signaling through Connexin 43 onto Mitochondrial ATP-Sensitive K+ Channels

Background: Ischemic preconditioning involves activation of signaling pathways, including activation of protein kinase C (PKC) and inhibition of glycogen synthase kinase 3beta (GSK3β). Opening of mitochondrial ATP-sensitive K+ (mitoKATP) channels has been proposed to play a critical role in ischemic preconditioning. Recently, we demonstrated that connexin 43 (Cx43) found in the inner mitochondrial membrane, is essential for cytoprotective signal transduction onto mitoKATP channels.Objectives: To dissect signal transduction targeted at mitoKATP channels and to possibly identify a functional link between mitoKATP channel activation, mitochondrial Cx43 and GSK3β activity.Methods: We used direct single-channel patch-clamp recordings of cardiac mitoplasts from C57/Bl6J mice and homozygous GSK3β-S9A mice with cardiac-specific expression of a constitutively active form of GSK3β, and performed Western blots and co-immunoprecipitation of isolated mitochondria.Results: MitoKATP channel activity was stimulated by the GSK3β small molecule inhibitor SB216763, which increased the open probability compared to control. The Cx43 inhibitor carbenoxolone and the Cx43 mimetic peptide 43GAP27 significantly reduced mitoKATP channel activation by SB216763, supporting the notion that GSK3 transfers cytoprotective signaling via Cx43 onto mitoKATP channels. In GSK3β-S9A mitoplasts mitoKATP currents could be recorded with similar single-channel properties as in wildtype mitochondria. Single-channel activation of mitoKATP by the PKC activator PMA was significantly attenuated in GSK3β-S9A mitoplasts. Western blot analysis revealed reduced phosphorylation of mitochondrial Cx43 at the PKC phosphorylation site Ser368 upon PMA application in mitochondria from GSK3β-S9A mice versus wildtype. Furthermore Immunoprecipitation of Cx43 from isolated mitochondria revealed a signal for GSK3β, while immunoprecipitation of GSK3β also showed a signal for Cx43, indicating an association of these proteins.Conclusions: PKC transmits a signal onto mitoKATP channels through GSK3β. This GSK3β signal transduction on mitoKATP channels is transferred via Cx43 within a multiprotein signaling module.

Are we ready for a new mechanism of action underlying digitalis toxicity?

It is said that the only certainties in life are death, taxes and tetrodotoxin. However, just when we think we understand the cellular mechanisms of a drug that has been used in the treatment of cardiac disease for over two centuries, along comes something completely different. Such is the case with the report in a recent issue of The Journal of Physiology by Ho et al. that cardiac glycoside (CG) toxicity in the form of spontaneous Ca2+ waves arises in response to altered function of cardiac ryanodine receptors (RyRs) induced by reactive oxygen species (ROS). The authors found that ROS generation oxidized thiol groups on RyRs, increasing their functional activity by increasing sensitivity to Ca2+ in the sarcoplasmic reticulum (SR) (Terentyev et al. 2008). The result is increased frequency of Ca2+ waves which, through sodium–calcium exchange (NCX), depolarize cardiac myocytes and induce triggered arrhythmias during CG toxicity. They also suggest that this effect of CGs occurs secondary to an interaction with (but not inhibition of) the Na+,K+-ATPase (NKA) that induces intracellular signalling through Src kinase, causing ROS production and opening of the mitochondrial ATP-dependent K+ channel (mito-KATP) (Tian et al. 2003; Pasdois et al. 2007). It is generally accepted that NKA inhibition by CGs causes both inotropic and toxic actions since the resulting intracellular Na+ accumulation, through NCX, also increases Ca2+. The raised Ca2+ increases contraction (resulting in positive inotropy) but eventually leads to toxicity in the form of Ca2+ waves (and triggered arrhythmias) as SR Ca2+ storage capacity is exceeded and Ca2+ overload develops. Since the 1960s, however, it has been known that different CGs have different abilities to produce both inotropy and toxicity, questioning the singular NKA hypothesis. One possible explanation involves direct CG actions on the RyR to increase its sensitivity to Ca2+ (Wasserstrom & Aistrup, 2005). Ho et al. suggest that an effect on RyRs might also occur indirectly, with ROS as the intermediary. CG binding to the NKA induces ROS generation through Src kinase signalling which does not involve increased Na+ and Ca2+. Rather, the authors suggest that NADPH oxidase causes release of mitochondrial ROS since increased ROS, RyR oxidation and Ca2+ wave frequency were reversed by inhibition of NADPH oxidase and mito-KATP channels but not by inhibition of xanthine oxidase. The authors are careful to point out that this mechanism may be distinct from the traditional mechanisms involving NKA inhibition and resulting intracellular Na2+ and Ca2+ changes as well as from potential direct actions on the RyR itself. Their results suggest a novel mechanism for the increased spontaneous Ca2+ release that underlies the toxic, arrhythmogenic effects of CGs and not their inotropic effects. Another interesting feature of their studies was the fact that they found that Ca2+ waves occurred despite a reduced SR Ca2+ load. Thus, RyR activation occurred at lower SR load, presumably because RyRs were sensitized to luminal Ca2+ after thiol modification. This is an unusual view of Ca2+ wave generation which ordinarily occurs through increased SR Ca2+ load which then increases RyR sensitivity, causing spontaneous release in the form of a wave. In contrast, Ho et al. present compelling evidence that CG induction of ROS with subsequent RyR modification increases functional activity despite a lower SR Ca2+ load. This mechanism may also provide a novel explanation for why toxicity might occur more easily with some CGs than with others; if one agent is more able to induce increased ROS than another, this might explain the differences in toxicity to therapeutic ratio that have been reported among different CGs for many years. An increase in RyR sensitivity to Ca2+, independent of changes in Na+ and Ca2+, suggests a unique mechanism for CG toxicity in the absence of increased SR load. Clinically, arrhythmias might then be susceptible to treatment with agents that reduce RyR sensitivity, obviating the use of generally ineffective conventional antiarrhythmic agents. Cardiac ROS suppression might also provide a fruitful therapeutic approach to treatment of CG toxicity. Consequently, CG effects on RyRs through a ROS intermediary might provide an entirely new and fresh view of how to deal therapeutically with the extremely common and highly dangerous effects of one of the world's most widely used medications for the treatment of heart disease.

Role of cadiocyte connexin 43 in mito-KATp channel mediated cardioprotection against ischemia-reperfusion injury induced by sevoflurane preconditioning in isolated rat hearts

Objective To evaluate the role of cadiocyte connexin 43 (Cx43) in mito-KATP channel mediated cardioprotection against ischemia-reperfusion(I/R) injury induced by sevoflurane preconditioning in isolated rat hearts.Methods Forty hearts from male adult SD rats weghing 200-250 g were excised and perfused in a Langendorff apparatus with K-H solution with 95 ％ O2-5 ％ CO2 at 36.5-37.5 ℃.Their hearts were randomly divided into 5 groups (n =8 each): control group (group C),group I/R,sevoflurane preconditioning group (group S),sevoflurane preconditioning + 5-HD group (group SH) and 5-HD group (group H).Myocardial I/R was induced by occlusion of the anterior descending branch of left coronary artery (LAD) for 30 min followed by 120 min reperfusion.After 10 min of equilibration,group C received continuous perfusion and LAD was exposed but not occluded.Group I/R received continuous perfusion for 30 min and LAD was occluded.In group S,SH and H,3％ sevoflurane,3 ％ sevoflurane mixed with 100μmmol/L 5-HD and 100 tmmol/L 5-HD was added into K-H solution to perfuse for 15 min respectively.After that,the hearts were washed by K-H solution for 15 min.HR,left ventricular systolic pressure ( LVSP),left ventricular diastolic pressure (LVDP) and ± dp/dtmax were recorded before administration (T0),immediately after administration (T1),immediately before ischemia ( T2 ),at 30 min of ischemia (T3) and 120 min of reperfusion (T4).At the end of reperfusion,left ventricular tissue was removed for determination of myocardial infarct size and expression of Cx43 and phosphor Cx43 (p-Cx43) by immunohistochemistry and Western blot respectively.Results Compared with group C,HR,LVSP and ± dp/dtmax were significantly decreased,LVDP was increased and the expression of Cx43 and p-Cx43 were down-regulated in groups I/R,SH and H (P ＜ 0.05).Compared with group I/R,HR,LVSP and ± dp/dtmax were significantly increased,LVDP and myocardial infarct size decreased,and the expression of Cx43 and p-Cx43 up-regulated in group S ( P ＜ 0.05),no significant difference was found in groups S + H and H( P ＞ 0.05 ).Conclusion Sevoflurane preconditioning can open mito-KATP channel,promote cadiocyte Cx43 phosphorylation,attenuate I/R injury in isolated rat hearts. Key words: Connexin 43 ; KATP channels; Anesthetics, inhalation; Ischemic preconditioning; Myocardial reperfusion injury

New Insights into the Mechanisms of Mitochondrial Preconditioning-Triggered Neuroprotection

Mitochondria fulfill a number of essential cellular functions, being recognized that the strict regulation of the structure, function and turnover of these organelles is an immutable control node for the maintenance of neuronal integrity and homeostasis. Many lines of evidence posit that mitochondria constitute a convergence point of preconditioning - a paradigm that affords robust brain tolerance in the face of neurodegenerative insults. Indeed, it has been described that preconditioning activates an adaptive reprogramming of mitochondrial biology in response to a noxious stress-stimulus, which in turn will contribute to augment both mitochondrial and neuronal tolerance. Mitochondrial reactive species (ROS), mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels and mitochondrial permeability transition pore have been identified as specific mitochondrial mediators and targets of the adaptive program underlying preconditioning. Recent studies further link mitochondrial biogenesis, dynamics and mitophagy to preconditioning, thereby representing novel mechanisms by which preconditioning may mediate brain tolerance. The present review summarizes the current views on how mitochondrial biology is linked to preconditioning-induced neuroprotection. A better understanding of the mitochondrial mechanisms underlying preconditioning will help in the development of novel therapeutic approaches with the primary goal of modulating mitochondria to enhance brain tolerance against neurodegenerative events.

Isoflurane decreases death of human embryonic stem cell-derived, transcriptional marker Nkx2.5+ cardiac progenitor cells

Cardiac progenitor cells (CPCs) derived from human embryonic stem cells (hESCs) can multiply and generate cardiomyocytes, offering their tremendous potential for cardiac regenerative therapy. However, poor survival under stressful conditions is a major hurdle in the regeneration. We investigated whether isoflurane-induced preconditioning can increase hESC-derived CPC survival under oxidative stress. Undifferentiated hESCs were cultured in suspension with 20% FBS (fetal bovine serum) and 20 ng/ml of BMP-4 (bone morphogenetic protein-4) to form embryoid bodies and grown onto Matrigel-coated plates for 2-3 weeks. To characterise the differentiated CPCs, immunostaining for Nkx2.5 (nonspecific transcriptional marker) and Isl-1 was performed. hESC-derived CPCs were exposed to oxidative stress induced by H(2) O(2) and FeSO(4) . For anaesthetic preconditioning, CPCs were exposed to isoflurane (0.25, 0.5, 1.0 mM). CPC survival was determined by trypan blue exclusion. A mitoK(ATP) channels inhibitor, 5-hydroxydecanoic acid (200 μM) and an opener, diazoxide (100 μM), were used to investigate the involvement of mitoK(ATP) channels. hESC-derived CPCs stained with Nkx2.5 were 95 ± 3% of total cell number. Isoflurane (0.5 and 1.0 mM)-preconditioned CPCs showed a significantly lower death rate compared with control (0.5 mM: 30.6 ± 10.7% and 1.0 mM: 28.5 ± 6.2% vs. control: 43.2 ± 9.9%). Inhibition of mitoK(ATP) channels with 5-HD completely abolished the protective effects of isoflurane. Diazoxide significantly decreased CPC death (29.5 ± 12.4%). However, when diazoxide was applied to CPC preconditioned with isoflurane, CPC death did not decrease further (28.7 ± 10.9%). Isoflurane increased hESC-derived Nkx2.5(+) CPC survival under oxidative stress, and mitoK(ATP) channels may be involved in the protective effect.

Possible involvement of caveolin in attenuation of cardioprotective effect of ischemic preconditioning in diabetic rat heart

BackgroundNitric oxide (NO) has been noted to produce ischemic preconditioning (IPC)-mediated cardioprotection. Caveolin is a negative regulator of NO, which inhibits endothelial nitric oxide synthase (eNOS) by making caveolin-eNOS complex. The expression of caveolin is increased during diabetes mellitus (DM). The present study was designed to investigate the involvement of caveolin in attenuation of the cardioprotective effect of IPC during DM in rat.MethodsExperimental DM was induced by single dose of streptozotocin (50 mg/Kg, i.p,) and animals were used for experiments four weeks later. Isolated heart was mounted on Langendorff's apparatus, and was subjected to 30 min of global ischemia and 120 min of reperfusion. IPC was given by four cycles of 5 min of ischemia and 5 min of reperfusion with Kreb's-Henseleit solution (K-H). Extent of injury was measured in terms of infarct size by triphenyltetrazolium chloride (TTC) staining, and release of lactate dehydrogenase (LDH) and creatin kinase-MB (CK-MB) in coronary effluent. The cardiac release of NO was noted by measuring the level of nitrite in coronary effluent.ResultsIPC- induced cardioprotection and release of NO was significantly decreased in diabetic rat heart. Pre-treatment of diabetic rat with daidzein (DDZ) a caveolin inhibitor (0.2 mg/Kg/s.c), for one week, significantly increased the release of NO and restored the attenuated cardioprotective effect of IPC. Also perfusion of sodium nitrite (10 μM/L), a precursor of NO, significantly restored the lost effect of IPC, similar to daidzein in diabetic rat. Administration of 5-hydroxy deaconate (5-HD), a mito KATP channel blocker, significantly abolished the observed IPC-induced cardioprotection in normal rat or daidzein and sodium nitrite perfused diabetic rat heart alone or in combination.ConclusionsThus, it is suggested that attenuation of the cardioprotection in diabetic heart may be due to decrease the IPC mediated release of NO in the diabetic myocardium, which may be due to up -regulation of caveolin and subsequently decreased activity of eNOS.

Noninvasive Delayed Limb Ischemic Preconditioning Attenuates Myocardial Ischemia-Reperfusion Injury in Rats by a Mitochondrial KATP Channel-Dependent Mechanism

We previously demonstrated in rats that noninvasive delayed limb ischemic preconditioning (LIPC) induced by three cycles of 5-min occlusion and 5-min reperfusion of the left hind limb per day for three days confers the same cardioprotective effect as local ischemic preconditioning of the heart, but the mechanism has not been studied in depth. The aim of this project was to test the hypothesis that delayed LIPC enhances myocardial antioxidative ability during ischemia-reperfusion by a mitochondrial K(ATP) channel (mito K(ATP))-dependent mechanism. Rats were randomized to five groups: ischemia-reperfusion (IR)-control group, myocardial ischemic preconditioning (MIPC) group, LIPC group, IR-5HD group and LIPC-5HD group. The MIPC group underwent local ischemic preconditioning induced by three cycles of 5-min occlusion and 5-min reperfusion of the left anterior descending coronary arteries. The LIPC and LIPC-5HD groups underwent LIPC induced by three cycles of 5-min occlusion and 5-min reperfusion of the left hind limb using a modified blood pressure aerocyst per day for three days. All rats were subjected to myocardial ischemia-reperfusion injury. The IR-5HD and LIPC-5HD groups received the mito K(ATP) channel blocker 5-hydroxydecanoate Na (5-HD) before and during the myocardial ischemia-reperfusion injury. Compared with the IR-control group, both the LIPC and MIPC groups showed an amelioration of ventricular arrhythmia, reduced myocardial infarct size, increased activities of total superoxide dismutase, manganese-superoxide dismutase (Mn-SOD) and glutathione peroxidase, increased expression of Mn-SOD mRNA and decreased xanthine oxidase activity and malondialdehyde concentration. These beneficial effects of LIPC were prevented by 5-HD. In conclusion, delayed LIPC offers similar cardioprotection as local IPC. These results support the hypothesis that the activation of mito K(ATP) channels enhances myocardial antioxidative ability during ischemia-reperfusion, thereby contributing, at least in part, to the anti-arrhythmic and anti-infarct effects of delayed LIPC.

Off‐Pump Glutamate Pre‐ and Post‐Conditioning against an Acute Myocardial Infarction: Role of ATP‐Sensitive Potassium Channels

Background Metabotropic glutamate receptor 5(mGluR5) is expressed in hearts. We determined whether infusion of glutamate (GLU) pre- (PreC) and post-conditions (Post-C) against acute myocardial infarction (MI) and if mitochondrial (Mito) KATP channel is involved (using 5-Hydroxydecanoate). Methods Anesthetized dogs (n = 46), instrumented to monitor LV contractility using sonomicrometry, subjected to 90 minutes LAD coronary artery occlusion and 4 hrs reperfusion (Rep). Dogs were infused with 50ml of vehicle (control), glutamate (Glu100μmol/L) or 5HD (5 mg/Kg)+GLU before ischemia (PreC) or before Rep (PostC). Coronary blood flow was measured using colored microspheres. LV function (stroke work/end diastolic relationships) was determined. The infarct size and LV and risk areas were measured. Four animals were excluded. Data were analyzed by ANOVA and presented as mean± SEM (n = 6). Results LV function significantly recovered in Glu-treated groups (p=NS vs., baseline, p < 0.05 vs. control, 5HD). Poor LV functional recovery was seen in the control (36.2±5%) and 5HD (46.1±6%)groups (p < 0.05 vs Glu groups). GLU reduced the infarct size (38.2±6% in the control group to11.1±4% and 14.3±3 in GLU PreC and PostC groups. 5-HD abolished Glu-infarct size limitation. Conclusions Infusion of GLU pre- and post-conditions the myocardium against INF. Mito-KATP channels are involved in GLU-mediated cardioprotection.

Effect of preconditioning with different concentrations of sevoflurane on hypoxia-reoxygenation-induced apoptosis in rat hippocampal neurons and the role of mitochondrial KATP channels

Objective To investigate the effect of preconditioning with different concentrations of sevoflurane on hypoxia-reoxygenation(H/R)-induced apoptosis in rat hippocampal neurons and the role of mitochondrial KATP(mito-KATP)channels.Methods Primary cultured hippocampal neurons isolated from newborn SD rats(＜24h)of both sexes,weighing 5-6 g,were randomly divided into 7 groups with 48 wells and 12 dishes in each one:control group(C group),H/R group,preconditioning with 6%,4%and 2% sevoflurane groups(S1-3 groups),5-hydroxydecanoate(5-HD,mito-KATP channel blocker)100 μmol/L preconditioning group(5-HD group)and preconditioning with 5-HD 100 μmol/L+6% sevoflurane group(5-HD+S group).The neurons were exposed to 4 h hypoxia followed by 24 h reoxygenation. In S1-3 groups, preconditioning was performed with 6% , 4% and 2% sevoflurane respectively before H/R. In 5-HD group, preconditioning was performed with 5-HD (final concentration 100 μmol/L) before H/R. In 5-HD + S group, preconditioning was performed with 5-HD 100 μmol/L and 6% sevoflurane before H/R. The neuronal viability, apoptosis rate and expression of Bcl-2 and Bax were determined after 24 h reoxygenation.Results The neuronal viability was significantly lower,while the apoptosis rate and expression of Bcl-2 and Bax were significantly higher in the other 6 groups than in group C(P＜0.01).The neuronal viability and expression of Bcl-2 were significantly higher,while the apoptosis rate and Bax expression were lower in S1-3 groups than in group H/R. There was no significant difference in the parameters mentioned above between 5-HD and 5-HD + S groups(P＞0.05).The neuronal viability and expression of Bcl-2 were significantly lower, while the apoptosis rate and Bax expression were higher in S2, S3 and 5-HD + S groups than in group S1, and in group S3 than in group S2(P＜0.0l) .Conclusion Sevoflurane preconditioning can inhibit H/R-induced apoptosis in rat hippocampal neurons and reduce the injury to neurons in a concentration-dependent manner, and the underlying mechanism may be related to activation of mito-KATP channels, up-regulation of Bcl-2 expression and down-regulation of Bax expression. Key words: Reperfusion injury; Oxygen; Apoptosis; KATP channels; Mitochondria; Neurons; Hippocampus; Sevoflurane

The Cardiac L-Type Ca 2+ Channel is Downregulated by Ischemic and Pharmacological Preconditioning

Ischemic preconditioning (IP) is a phenomenon in which briefs periods of ischemia activate endogenous mechanisms that protect cardiomyocytes from subsequent ischemia. Pharmacological Preconditioning (PPC) with mitochondrial ATP-sensitive K+ channel (mitoKATP) openers such as diazoxide mimics IP. However, changes in Ca2+ homeostasis during IP and PPC, particularly in Ca2+ channel activity, are poorly understood. We investigated the effects of IP and PPC on cardiac L-type Ca2+ channels.IP was achieved by four cycles of ischemia-reperfusion in isolated hearts. PPC was induced in isolated hearts and in dissociated cardiomyocytes from adult rats by preincubation withdiazoxide. We measured reactive oxygen species (ROS) production and Ca2+ signals associated with action potentials usingfluorescent probes. L-type currents were measured with the whole-cell patch-clamp technique. Levels of the alpha1c subunit of L-type channelsin the cellular membrane were measured by Western blot.IP produced a 20% reduction in alpha1c subunit levels. PPC was accompanied by a 50% reduction in alpha1c subunit levels, and by a reversible fall in L-type current amplitude and Ca2+transients. These effects were prevented by the ROS scavenger N-acetyl-L-cysteine (NAC) or by the mitoKATP channel blocker5-hydroxydecanoate (5-HD). PPC significantly reduced infarct size, an effect blocked by NAC and 5-HD. Nifedipine alsoconferred protection against infarction when applied during the reperfusion period. Downregulation of the alpha1c subunit andCa2+ channel function were prevented in part by the protease inhibitor leupeptin.We conclude that IP andPPC downregulate the alpha1c subunit by a proteolytic process in which ROS are involved. This in turn leads to a reduced Ca2+ influx and attenuates Ca2+ overload during reperfusion.Grants: CONACYT: 60880 (J.S) and 102100 (M.C.G).