Articles in PresS. Am J Physiol Heart Circ Physiol (August 26, 2005). doi:10.1152/ajpheart.00763.2005
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Cardiac mitochondrial preconditioning by big Ca2+-sensitive K+ channel opening requires superoxide radical generation David F. Stowe,1-5 Mohammed Aldakkak,1 Amadou K.S. Camara,1 Matthias L. Riess,1 Andre Heinen,1 Srinivasan G. Varadarajan,1 Ming-Tao Jiang1
Anesthesiology Research Laboratories, Departments of Anesthesiology1 and Physiology,2 Cardiovascular Research Center,3 The Medical College of Wisconsin, Milwaukee, Wisconsin 53226, VA Medical Center Research Service,4 Milwaukee, Wisconsin 53295, and Department of Biomedical Engineering,5 Marquette University, Milwaukee, Wisconsin 53233
This work has been published in part in abstract form: Jiang MT et al: Biophys J 86 (suppl.);465A;2004. Stowe DF et al: FASEB J 18:670.4;2004. Stowe DF et al: FASEB J 19:386.26;2005.
Please address all correspondence to: David F. Stowe, M.D., Ph.D., M4280, 8701 Watertown Plank Road, Medical College of Wisconsin, Milwaukee, Wisconsin 53226. Tel: 414-456-5722, Fax: 414456-6507, email:
[email protected]
Abbreviated title: cardiac preconditioning and mitochondrial BKCa channels
Subject code list: cell signaling; ischemic biology; oxidant stress; energy metabolism
Copyright © 2005 by the American Physiological Society.
2 ABSTRACT ATP –sensitive K+ (KATP) channel opening in inner mitochondrial membranes (IMM) protects hearts from ischemia reperfusion (IR) injury. Opening of the “big” conductance Ca2+ –sensitive K+ channel, BKCa, is now also known to elicit cardiac preconditioning. We investigated the role of pharmacologic opening of the BKCa channel on inducing mitochondrial preconditioning during ischemia and reperfusion and the role of O2 derived free radicals in modulating protection by putative mBKCa channel opening. Left ventricular pressure (LVP) was measured with a balloon and transducer in guinea pig hearts isolated and perfused at constant pressure. NADH, reactive O2 species (ROS), principally superoxide (O2•-), and mitochondrial m[Ca2+] were measured spectrophotofluorometrically at the LV free wall using autofluorescence and fluorescent dyes dihydroethidium and indo 1, respectively. BKCa channel opener NS1619 (NS) was given for 15 min ending 25 min before 30 min of global IR. Either MnTBAP (TB), a synthetic dismutator of O2•-, or an antagonist of the BKCa channel, paxilline (PX), was given alone or for 5 min before, during and 5 min after NS. NS pretreatment resulted in a 2.5 fold increase in developed LVP and a 2.5 fold decrease in infarct size. This was accompanied by less O2•-, generation, decreased m[Ca2+], and more normalized NADH during early ischemia and throughout reperfusion. Both TB and PX antagonized each preconditioning effect. This indicates 1) NS induces a mitochondrial -preconditioned state evident during early ischemia, presumably on mBKCa channels, 2) NS effects are blocked by BKCa antagonist PX, and 3) NS –induced preconditioning is dependent on production of ROS. Thus, NS may induce mitochondrial ROS release to initiate preconditioning.
Index terms: calcium sensitive K+ channel, heart, mitochondria, reactive oxygen species, redox balance
3 INTRODUCTION Depressed mitochondrial bioenergetics with excess reactive oxygen species (ROS) generation and mitochondrial (m) Ca2+ loading are major factors underlying ischemia/reperfusion (IR) injury. Prophylactic measures to reduce cardiac IR injury include ischemic preconditioning (IPC, i.e., brief pulses of ischemia and reperfusion before longer ischemia) and pharmacologic preconditioning (PPC), i.e., cardiac protection elicited some time after a drug is washed out. PPC is theoretically a better approach because it does not require brief ischemia. A few exogenous drugs, e.g. pinacidil and diazoxide that induce PPC, possibly act on a ATP –sensitive K+ channel (KATP). It would appear likely that the opening of other mitochondrial potassium channels would also elicit PPC. The membranes of vascular smooth muscle, neural, and secretory cells contain large or “big”conductance (100-300 pS) K+ channels (BKCa) activated by increased [Ca2+]i and by cell membrane depolarization (4). BKCa channel opening allows cytosolic K+ efflux, which promotes cell membrane repolarization; this in turn reduces Ca2+ entry by closing voltage-dependent Ca2+ channels (40, 41, 45). Siemen et al. (35) first demonstrated BKCa channels in the inner mitochondrial membrane (IMM) in a glioma cell line. Xu et al. (42) reported NS1619 –induced activation of BKCa channels isolated to IMM of guinea pig cardiac myocytes; they also reported that NS1619 protected against global IR injury in rabbit isolated hearts. Although they continuously infused NS up to the onset of ischemia, this led to the possibility that drugs that open mBKCa, like mKATP, channels, also induce cardiac PPC via a ‘memory’ effect. More recently others have shown that NS1619 could precondition isolated mice (39) and rat (7) hearts subjected to global ischemia, and in situ dog hearts subjected to regional ischemia (34). NS1619 -induced effects were attenuated or blocked by charybdotoxin and iberiotoxin (34) or by paxilline (PX) (7, 39), an antagonist of BKCa channel opening (32, 42). The mechanism for PPC -induced protection by BKCa channel opening is not known. We hypothesized that Ca2+ –sensitive K+ channel opening within the IMM initiates a mitochondrial protective effect evidenced by attenuated changes in several indices of mitochondrial function during and after ischemia, as well as improved myocardial contractile and vascular function, and reduced infarct size on reperfusion after ischemia. To examine this we infused and washed out NS before ischemia and examined specifically the effect of NS on attenuating mitochondrial dysfunction during IR by near continuous measurement of NADH, ROS (superoxide, O2•-) and m[Ca2+] in isolated hearts. We gave PX to verify its effect to antagonize NS. Because protective effects of putative KATP channel openers can be abolished by ROS scavengers (28), we also bracketed NS with a dismutator of O2•-,
4 MnTBAP (TB), to assess if NS –induced mBKCa opening. requires O2•- , presumably generated within the mitochondrial respiratory complex, to initiate PPC. METHODS Langendorff heart model. The investigation conformed to the Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23, revised 1996). Guinea pig hearts were isolated and prepared as described in detail (2, 6, 17, 18, 29-31) with care to minimize IPC. These were preoxygenation, maintaining respiration after anesthesia with ketamine (50 mg/kg), and immediately perfusing the aortic root with cold solution. Hearts were instrumented with a saline filled balloon and transducer to measure left ventricular pressure (LVP) and an aortic flow probe to measure coronary flow (CF). Heart rate and rhythm were measured via atrial and ventricular electrodes. Hearts were perfused in Langendorff mode at 55 mmHg with modified Krebs-Ringer’s solution at 37°C. Heart rate (HR) and rhythm, myocardial function (isovolumetric LVP) and coronary flow were measured continuously. At 120 min reperfusion, hearts were stained with 2,3,5 –triphenyltetrazolium chloride (TTC) and infarct size was determined as a percentage of ventricular heart weight (2). Cardiac measurements. Either NADH, m[Ca2+], or ROS (primarily O2•-) were measured near continuously from the LV using one of three excitation and emission fluorescence spectra (2, 6, 17, 18, 29, 31) in different subsets of hearts. A trifurcated fiberoptic probe (3.8 mm2 per bundle) was placed against the LV to excite and record light signals at specific wavelengths using spectrophotofluorometers (SLM Amico-Bowman and Photon Technology International). NADH autofluorescence was assessed at 350 nm excitation and 450/390 nm emission wavelengths. Alternatively, hearts were loaded with 6 µM indo 1 AM (load 30 min, washout 20 min) and Ca2+ transients were recorded using the excitation wavelength for NADH with emissions recorded at 390 and 450 nm. After initially recording Ca2+ transients, hearts were perfused for 15 min with 100 µM MnCl2 to quench cytosolic Ca2+ to reveal primarily [mCa2+] (29). In other hearts, as described earlier (6, 17, 18), dihydroethidium (DHE, 10 µM) was loaded for 20 min and washed out for 20 min. The LV wall was excited at 540 nm and emitted light recorded at 590 nm to measure a labile fluorescence signal that is primarily a marker of the free radical O2•- (37, 44). DHE enters cells and is oxidized by O2•-, which converts it to a reversible ethidium-like compound which causes a red-shift in the EM light spectrum (44). Myocardial fluorescence intensity was recorded in arbitrary fluorescence units, afu, over 12 s each min at a 100 µs/s sampling rate throughout experiments for DHE, and over 2.5 s at a 100 µs/s sampling rate during 35 discrete sampling periods throughout each experiment for NADH and m[Ca2+]. For each
5 fluorescence study none of the drugs alone had any appreciable effect on autofluorescence. m[Ca2+] was corrected for underlying changes in NADH autofluorescence during ischemia and reperfusion for each group. Each signal was digitized and recorded at 200 Hz (Power LAB/16sp, Chart and Scope 3.6.3, ADInstruments) on G4 Macintosh computers for later analysis using specifically designed programs (MATLAB, MathWorks and Microsoft Excel) software. All variables were averaged over the 2.5 or 12 s sampling period. Protocol. Hearts were infused with 3 µM NS1619 (1-(2’-hydroxy-5’-trifluoromethylphenyl)-5trifluoromethyl-2(3H) benzimid-axolone) for 15 min ending 25 min before the onset of 30 min global ischemia. In some hearts NS was bracketed with 1 µM paxilline (PX), a blocker of the BKCa channel (32), or 20 µM TB (Mn(III) tetrakis (4-benzoic acid) porphyrin), a dismutator of O2•-. PX or TB was given for 5 min before starting NS, during NS, and for 5 min after stopping NS. Reperfusion lasted 120 min. Additional studies (not displayed) showed that PX or TB given alone as a pre-treatment, i.e., without NS, when compared to drug-free controls, had no effect on any of the variables measured. Statistical analyses. A total of 126 isolated hearts experiments were divided into 6 groups: control, NS, NS+PX, NS+TB, PX and TB. NADH was measured in 6-8 hearts per group, ROS in 6-8 hearts per group, and m[Ca2+] in 6-8 hearts per group. LVP and coronary flow were measured in all hearts so that there were 21 hearts per group for functional data. Infarct size was measured in a blinded manner in 7 hearts of each group. All data were expressed as means ± standard error of means. Appropriate comparisons were made among groups that differed by a variable at a given condition or time, and within a group over time compared to the initial control data. Statistical differences were measured across groups at specific time points (20, 50, 80, 100, 130, and 240 min). Differences among variables were determined by 2 way multiple analysis of variance for repeated measures (Statview® and CLR anova® software; Macintosh® computers); if F tests were significant, appropriate post-hoc tests (Student-Newman-Keul’s, or Duncan’s) were used to compare means. Mean values were considered significant at P values (two-tailed)
