Endogenous and agonist-induced opening of mitochondrial big vs. small Ca²⁺-sensitive K⁺ channels on cardiac cell and mitochondrial protection

Abstract

Both big (BK_Ca) and small (SK_Ca) conductance Ca²⁺-sensitive K⁺ channels are present in mammalian cardiac cell mitochondria (m). We used pharmacological agonists and antagonists of BK_Ca and SK_Ca channels to examine the importance of endogenous opening of these channels and the relative contribution of either or both of these channels to protect against contractile dysfunction and reduce infarct size after ischemia reperfusion (IR) injury through a mitochondrial protective mechanism. Following global cardiac IR injury of ex vivo perfused guinea pig hearts we found the following: both agonists NS1619 (for BK_Ca) and DCEB (for SK_Ca) improved contractility; BK_Ca antagonist paxilline (PAX) alone or with SK_Ca antagonist NS8593 worsened contractility and enhanced infarct size; both antagonists PAX and NS8593 obliterated protection by their respective agonists; BK_Ca and SK_Ca antagonists did not block protection afforded by SK_Ca and BK_Ca agonists, respectively; and all protective effects by the agonists were blocked by scavenging superoxide anions (O₂^•−) with TBAP. Contractile function was inversely associated with global infarct size. In in vivo rats, infusion of NS8593, PAX, or both antagonists enhanced regional infarct size while infusion of either NS1619 or DCEB reduced infarct size. In cardiac mitochondria isolated from ex vivo hearts after IR, combined SK_Ca and BK_Ca agonists improved respiratory control index (RCI) and Ca²⁺ retention capacity (CRC) compared to IR alone, whereas the combined antagonists did not alter RCI but worsened CRC. Although the differential protective bioenergetics effects of endogenous or exogenous BK_Ca and SK_Ca channel opening remain unclear, each channel likely responds to different sensing Ca²⁺ concentrations and voltage gradients over time during oxidative stress-induced injury to individually or together protect cardiac mitochondria and myocytes.

Introduction

Altered mitochondrial (m) bioenergetics, excess reactive oxygen species (ROS) emission, and excess mCa²⁺ overload, are major factors that underlie ischemia and reperfusion (IR) injury.¹ Prophylactic measures targeted to mitochondria that reduce cardiac IR injury^{2, 3} include ischemic preconditioning (IPC, i.e., brief pulses of ischemia and reperfusion before longer index ischemia) and pharmacologic pre-conditioning (PPC), i.e., cardiac protection elicited some time after the drug is washed out before index IR. Drugs that reduce cardiac metabolism or mitochondrial bioenergetics when given before and or after IR also afford protection.

The induction of mitochondrial (m) K⁺ influx is now recognized as an important underlying mechanism to reduce IR injury. We reported previously that activation by NS1619 of the large (big) conductance Ca²⁺-sensitive K⁺ channel (BK_Ca, K_Ca1.1_, maxi-K), that is found in the cardiac myocyte inner mitochondrial membrane (IMM), induced pharmacologic preconditioning (PPC) and that this protection was blocked by the mitochondrial targeted superoxide anion (O₂^•−) scavenger TBAP.⁴ We subsequently reported that NS1619 had biphasic effects on mitochondrial respiration, membrane potential (ΔΨ_m), and O₂^•− emission in isolated mitochondria.^{5, 6} Due to our findings and many other reports on the protective effects of BK_Ca and putative K_ATP channel agonists in mitochondria, it is likely that opening of other mK⁺ channels might also be protective but rely on different ligands and conditions for activation.

K_Ca channels of intermediate or small conductance were first identified in non-cardiac cells^7–10 as membrane bound, calmodulin (CaM)-dependent and activated by Ca²⁺. These channels have smaller unit conductances of 3–30 (small, SK_Ca) and 20–90 (intermediate IK_Ca) pS.¹¹ The opening of SK_Ca channels is initiated by Ca²⁺ binding to calmodulin within the C terminus of the channel to form a dimer.^{12, 13} Of the four known genes encoding SK_Ca channel proteins, one of these, the K_Ca2.3 (aka SK3) isoform, was found in vascular endothelial cell membranes where it exerted a potent, tonic hyperpolarization that promoted reduced vascular smooth muscle tone.¹⁴ Later, the isoform K_Ca2.2 (aka SK2) was found in rat and human heart cell sarcolemma membranes by Western blot and RT-PCR.¹⁵ Amplification of the channel from total atrial and ventricular RNA showed much greater expression in atria than in ventricles, and electrophysiological recordings exhibited much greater atrial than ventricular sensitivity to AP repolarization by apamin, a selective SK_Ca antagonist.^{15, 16}

Dolga et al.¹⁷ found that the SK2 channel was expressed also in mitochondria of neuronal HT-22 cells. At the same time, we furnished the first evidence for the presence of SK2 and SK3 isoforms in the IMM of guinea pig cardiac mitochondria.¹⁸ We tested the functionality of SK_Ca channel opening¹⁸ by perfusing isolated hearts transiently before ischemia with the K_Ca3.1 (IK_Ca1)^{7, 9, 19} and K_Ca2.2 and K_Ca2.3^20–23 opener DCEBIO (DCEB). We found¹⁸ that DCEB elicited PPC in a manner similar to that of the BK_Ca channel agonist NS1619.⁴ Either the SK_Ca antagonist NS8593^{24, 25} or TBAP, which dismutates O₂^•− to H₂O₂, abolished the cardioprotection.¹⁸ DCEB also reduced the deleterious effects of IR injury on mitochondrial bioenergetics by attenuating cardiac mitochondrial Ca²⁺ overload and O₂^•− levels, and by preserving (more reduced redox state) cardiac mitochondrial NADH and FAD levels after IR.¹⁸ These studies established that K_Ca channel opening initiates a cascade of O₂^•−-dependent cardiac protective mechanisms that are based primarily in cardiac myocyte mitochondria. In a recent extension of this research,²⁶ we: a) confirmed SK_Ca protein localization in cardiac ventricular mitochondria of rats and humans, in addition to guinea pigs; b) identified mitochondrial splice variants of SK3 in guinea pig and human cardiac mitochondria; and c) found that overexpression or silencing of SK3 reduced or enhanced, respectively, stress induced damage in two cardiac cell lines, HL-1 and H9c2.

Our aim in the present study was to determine if endogenous opening of the BK_Ca and or SK_Ca channel protected hearts and mitochondria from IR injury and if there were differential or additive cardioprotective effects of drug-induced opening of BK_Ca and SK_Ca channels. To test this we: a) conducted a pharmacological comparison of SK_Ca and BK_Ca agonists and antagonists given before and after ischemia on global ventricular infarct size and developed left ventricular pressure (LVP) in isolated ex vivo perfused guinea pig hearts; b) examined the effect of SK_Ca and BK_Ca agonists and antagonists on regional infarct size when infused in rats in vivo; c) assessed the role of mitochondrial-targeted O₂^•− scavenging by TBAP to block protection by the two agonists; and d) measured respiratory control index (RCI) and Ca²⁺ retention capacity (CRC) after IR injury to further assess the likely role of mitochondria in protecting or impairing cardiac function and damage by treating ex vivo perfused hearts with SK_Ca and BK_Ca agonists or antagonists before and after IR injury.

Materials and methods

Function and Infarction in Ex Vivo Guinea Pig Hearts after Global IR Injury

The investigation conformed to the Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23, revised 1996); all animal protocols were reviewed and approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee. The hearts of adult guinea pigs of either sex were isolated and prepared as described previously in detail.^{4, 18, 27–33} Guinea pig hearts were chosen because of their more human-like cardiac action potentials and ion currents compared to rats.³⁴ In brief, animals were injected intraperitoneally with heparin (1000 units) and ketamine (50 mg/kg). Once anesthetized, thoracotomy was performed, hearts were excised, and their aortas immediately perfused in retrograde manner with a cold modified Krebs-Ringer’s (KR) perfusate. After aortic cannulation, hearts were transferred to a Langendorff perfusion setup and instrumented with a saline filled balloon and transducer to measure isovolumetric, developed (systolic-diastolic) LVP and perfused at a constant aortic root pressure of 55 mmHg with the KR solution at 37°C. Heart rate was measured from right atrial and ventricular recording electrodes, and coronary (aortic) flow was measured with a flow transducer. Hearts were subjected to 35 min global ischemia and 120 min reperfusion; hearts not used for mitochondrial isolation were stained with 2,3,5-triphenyltetrazolium chloride (TTC) and infarct size was determined as a percentage of total ventricular heart weight.^{4, 18, 28, 32} Infarct size measurements were blinded to the drug administered.

Isolated guinea pig hearts (18 groups of 6–8 hearts each) were perfused with no drugs (control) or test drugs, i.e. BK_Ca or SK_Ca agonists or antagonists, or TBAP alone or in combination, before and after the 35 min period of global ischemia. Time control (TC) experiments were also conducted, i.e., perfusion of isolated hearts with vehicle (DMSO) but without drugs or IR for the duration of the IR protocol. Time control (TC, no IR) functional variables decreased less than 5% over a 2.5 h period. To assess endogenous cardioprotection mediated by IR-induced BK_Ca and SK_Ca channel activation, antagonists were given beginning 20 min before ischemia. To assess exogenous BK_Ca and or SK_Ca activation, agonists were given beginning 15 min before ischemia; all drugs were discontinued 20 min after the onset of reperfusion. Functional variables for display in figures were recorded at 25 min before ischemia (baseline) and at 120 min reperfusion. Concentrations of drugs were gauged to elicit maximal effects as reported in the literature.^35–38 Drugs were dissolved in less than 0.1% DMSO in KR buffer (vehicle) and perfused into isolated hearts. They were: 3 μM DCEBIO (DCEB), a non-selective K_Ca2.2 and K_Ca2.3 channel agonist;^{7, 20–23} 10 μM NS8593, a specific antagonist of SK_Ca channels;^{24, 25} 3 μM NS1619, an agonist of BK_Ca channels; 40 μM paxilline (PAX), an antagonist of BK_Ca channels,³⁹ and 20 μM TBAP, a chemical dismutator of O₂^•− that preferentially enters the mitochondrial matrix to convert O₂^•− to H₂O₂, which is eventually converted to H₂O. NS8593 was selected as a specific antagonist of SK_Ca channels because DCEB can also open IK_Ca channels.^{7, 9, 19, 23} Generally, combinations of drugs would include an agonist with an antagonist, both agonists, both antagonists, or TBAP with either or both agonists. The protocols were designed to expose any protection afforded by endogenous BK_Ca vs. SK_Ca channel activation and any potential additive or potentiating cardiac protective or anti-protective effects of drug-induced opening or blocking of BK_Ca and SK_Ca channels.

Infarction in In Vivo Rat Hearts after Regional IR Injury

Adult rats (250–300 g) of either sex were anesthetized with inactin 150 mg/kg i.p., intubated, and artificial respiration was initiated. The right carotid artery was cannulated to monitor blood pressure continuously. After thoracotomy a suture was placed around the proximal LAD to induce reversible coronary artery occlusion verified by regional epicardial cyanosis. Rats were chosen because the area-at-risk for cardiac infarction is more uniform than in guinea pigs.⁴⁰ Hearts (6 groups of 4 each) were subjected to 35 min LAD occlusion by tightening the suture; releasing the suture was followed by 120 min reperfusion. Other hearts had the suture placed around the LAD but not tightened (sham, time control, (TC)). Inactin, 25 mg/kg i.p., was supplemented as needed. Infarct size was assessed after reperfusion using the Evan’s Blue area-at-risk and TTC staining techniques and expressed as percent ventricular area-at-risk. Drugs, dissolved in 0.1% DMSO and infused at 0.35 mL/h via the left jugular vein, were saline + 0.1% DMSO (vehicle control) or DCEB (30 μg/kg/min), NS1619 (30 μg/kg/min), NS8593 (100 μg/kg/min), or PAX (20 μg/kg/min). Total DMSO infused was less than 250 μg/h. Drug concentrations for infusion were obtained from the literature.^35–38 PAX and NS8593, the antagonists for the BK_Ca and SK_Ca channels, respectively, were also perfused together to assess for any additive damaging effects. Drugs were infused 20 min before regional ischemia and for 20 min after release of the LAD occlusion. Infarct size measurements were blinded to the drug administered.

Respiration and Ca²⁺ Retention Capacity in Mitochondria Isolated after Global IR Injury

Ex vivo guinea pig hearts were subjected to global ischemia with or without drug treatment as described above. At 20 min of reperfusion some hearts (6 groups of approximately 5 each) that were not used to measure infarct size were removed from the perfusion apparatus and the ventricles were minced in cold mitochondrial isolation buffer on ice. Mitochondria were isolated by differential centrifugation as described previously^{5, 6, 41–46} and the mitochondria (0.5 mg protein/mL) were suspended in respiration buffer containing 130 mM KCl, 5 mM K₂HPO₄, 20 mM MOPS, 40 μM EGTA (carried over from the isolation buffer), 1 μM Na₄P₂O₇, 0.1% BSA, pH 7.15 adjusted with KOH. To test mitochondrial viability and function, the respiratory control index (RCI, state 3/state 4) was determined under different substrate conditions: Na-pyruvate (P, 10 mM) or Na-succinate (S, 10 mM) or S + rotenone (R, 4 μM). State 3 respiration was determined after adding 250 μM ADP. In other experiments using the same mitochondrial pellet, Ca²⁺ retention capacity (CRC) was evaluated by measuring the decline in buffer [Ca²⁺] with Fura-4 penta-K⁺ using fluorescence spectrophotometry (QM-8, Horiba/Photon Technology International, PTI) as described previously^44–48 during bolus additions of 20 μM CaCl₂ every 90 s. Based on the similar improvement in cardiac function by individual or combined agonists, only the mitochondrial effects of the combined agonists NS1619 and DCEB were examined in the respiration and CRC experiments.

Statistical Analyses of Heart and Mitochondrial Data

Data obtained from hearts and mitochondria 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. Statistical differences were measured across groups using data comparisons at 25 min before global or regional ischemia vs. 120 of reperfusion (ex vivo and in vivo hearts). Mitochondria were isolated 20 min after reperfusion and data were collected within 2 h of isolation. Differences among variables were determined by two-way multiple ANOVA for repeated measures (Statview^® and CLR anova^® software programs for Macintosh^®); if F tests were significant, appropriate post-hoc tests (e.g., Student-Newman-Keul’s, SNK) were used to compare means. Mean values were considered significant at P values (two-tailed) <0.05.

Results

Global Protection by BK_Ca and SK_Ca Agonists and Enhanced Damage by BK_Ca plus SK_Ca Antagonists after Cardiac IR Injury in Ex Vivo Guinea Pig Isolated Hearts

We conducted an extensive analyses of the interactions of SK_Ca and BK_Ca agonists and antagonists on cardiac function and tissue damage based on assessment in changes in developed LVP (Fig. 1A–F) and % infarct size (Fig. 2A–F) after perfusion of hearts with single or combined drugs. Developed LVP was markedly decreased after 35 min global ischemia and 120 min reperfusion compared to TC (Fig. 1, IR) whereas % infarct size was markedly increased (Fig. 2, IR). After perfusion of hearts with the agonists and antagonists we found that: a) DCEB or NS1619 alone was equally protective as was combining NS1619 + DCEB on developed LVP (Fig. 1A) and infarct size (Fig. 2A); b) NS8593 alone did not worsen cardiac function (Fig. 1B) or infarct size (Fig. 2B) when compared to IR injury alone, whereas PAX alone and PAX + NS8593 further worsened cardiac function and infarct size; c) protection by NS1619 (Figs. 1C, 2C) was blocked by PAX and PAX+NS8593 but not by NS8593 alone; d) protection by DCEB (Fig. 1D, Fig. 2D) was blocked by NS8593 but not altered by PAX alone or by NS1619 + PAX; e) TBAP completely blocked protection induced either by NS1619 or DCEB or combined NS1619 + DCEB (Figs. 1E, 2E). In general, the effects of the BK_Ca and SK_Ca agonists and antagonists on developed LVP (Fig. 1) were approximately inversely proportional to their effects on infarct size (Fig. 2). Spontaneous heart rate before ischemia was approximately 240 beats/min in TCs and before ischemia. Heart rate, LVP and infarct size were not affected by perfusion of any of the drugs before IR injury; heart rate and coronary flow returned to control values during the reperfusion phase in all groups.

Fig. 1.

Average developed (systolic-diastolic) left ventricular pressure (LVP) 120 min after global IR injury when isolated guinea pig hearts were perfused without ischemia (Time Controls, TC); with 35 min ischemia and 120 min reperfusion (IR); or with IR + BK_Ca and or SK_Ca channel agonists (A); IR + BK_Ca and or SK_Ca channel antagonists (B); IR + BK_Ca agonist and or BK_Ca, SK_Ca antagonists (C); IR + BK_Ca agonist and or SK_Ca or BK_Ca antagonist (D); and SOD dismutator + BK_Ca and or SK_Ca agonist (E). Developed LVP over time for the IR only, IR + SK_Ca agonist and SK_Ca + BK_Ca agonist groups are displayed (F). Note the worsened effects on developed LVP of BK_Ca and BK_Ca + SK_Ca antagonists (green bars) vs. IR only (red bars); the block of protection by the BK_Ca or SK_Ca agonists when the BK_Ca or SK_Ca antagonists, respectively, was present (green bars); the maintained protection by the BK_Ca and or SK_Ca agonists in the presence of either the SK_Ca or the BK_Ca antagonist, respectively, (blue bars); and the loss of protection by the SK_Ca and or BK_Ca agonists in the presence of SOD inhibition by TBAP (orange/green bars). For each treatment group n = 5–6 hearts; note that for 32 of these hearts’ mitochondria were isolated after 20 min reperfusion to assess RCI and CRC (Figs. 4,5). Data expressed as mean ± sem. *^,§ P<0.05.

Fig. 2.

Percent infarct size assessed 120 min after global IR injury when the same (Fig. 1) isolated guinea pig hearts were perfused without ischemia (Time Controls, TC); with 35 min ischemia and 120 min reperfusion (IR); or with IR + BK_Ca and or SK_Ca channel agonists (A); IR + BK_Ca and or SK_Ca channel antagonists (B); IR + BK_Ca agonist and or BK_Ca, SK_Ca antagonists (C); IR + BK_Ca agonist and or SK_Ca or BK_Ca antagonist (D); and SOD dismutator + BK_Ca and or SK_Ca agonist (E). Representative hearts from 4 groups displaying normal and infarcted tissue are displayed (F). Note the enhanced infarct size after treatment with BK_Ca or BK_Ca + SK_Ca antagonists (green bars) vs. IR only (red bars); the block of tissue protection by the BK_Ca or SK_Ca agonists when the BK_Ca or SK_Ca antagonists, respectively, was present (green bars); the maintained reduction in infarct size by the BK_Ca and or SK_Ca agonists in the presence of either the SK_Ca or the BK_Ca antagonist, respectively, (blue bars); and the loss of protection by the SK_Ca and or BK_Ca agonists in the presence of SOD inhibition by TBAP (orange/green bars). For each treatment and control group n=4. *^,§P<0.05.

Tissue Protection by BK_Ca and SK_Ca Agonists and Enhanced Tissue Damage by BK_Ca plus SK_Ca Antagonists after Regional IR Injury in in Vivo Rat Hearts

We then tested if the BK_Ca and SK_Ca agonists and antagonists had any effects on the heart when delivered intravenously before ischemia and during reperfusion. Rats were infused with vehicle, PAX, NS8593, or PAX+NS8593, or with NS1619 or DCEB, before and after regional ischemia (LAD occlusion). Infarct size (% area-at-risk) was assessed after 120 min reperfusion (Fig. 3). There was no significant infarction in the sham animals and the infusion of drugs did not alter LVP or heart rate (data not shown) before IR. NS8593 or PAX infused alone enhanced infarct size whereas simultaneous infusion of both NS8593 and PAX additionally enhanced infarct size; infusion of either NS1619 or DCEB reduced infarct size.

Fig. 3.

Infarct size (% area-at-risk) without (time control, TC) or after regional ischemia (LAD occlusion, IR) when 28 intact rats were infused i.v. with no drug (IR only), or with IR plus BK_Ca and or SK_Ca channel antagonists, or with the BK_Ca or SK_Ca channel agonist. Note that treatment either with the SK_Ca or the BK_Ca channel antagonist (green bars), or both together, increased infarct size vs. IR only (red bar), and that treatment with either the BK_Ca or SK_Ca agonist reduced infarct size (blue bars) vs. IR only. Average left ventricular area-at-risk was 39.2±2.1. For each treatment and control (sham) group n=4. *^,§P<0.05.

Overall, these heart experiments demonstrated endogenous SK_Ca and BK_Ca channel activation in vivo rat hearts based on their antagonists’ effects to enhance regional infarct size as well as endogenous BK_Ca channel activation in ex vivo guinea pig hearts based on an additive effect of inhibiting both channels on contractile function. The experiments also established the selectivity of the respective antagonists to block their respective agonists, and the role of ROS in mediating the exogenous protection afforded by both BK_Ca and SK_Ca channel agonists.

Mitochondrial Respiration and mCa²⁺ Uptake after IR injury in Guinea Pig Ex Vivo Hearts Treated with BK_Ca and SK_Ca Agonists or Antagonists

Cardiac protection due to BK_Ca and SK_Ca channel opening may be mediated largely by effects on mitochondrial bioenergetics. Thus we tested if combined BK_Ca and SK_Ca agonists improved oxidative-phosphorylation assessed by respiration rates and RCI in cardiac mitochondria isolated 20 min after reperfusion and 35 min global ischemia (Table 1 and Fig. 4). The agonists were given together because we observed equivalent effects with the single agonists in the hearts and because the agonists given together did not additionally improve cardiac function or reduce infarct size when compared to IR control (Figs. 1–3). Representative traces of O₂ consumption (respiratory rate) in mitochondria energized with pyruvate (Fig. 4A upper left) are shown with mean RCI’s under the three substrate conditions (Fig. 4A). Under each substrate condition, states 2 and 4 respiration (Table 1) were increased, and state 3 respiration was decreased in mitochondria isolated after 20 min reperfusion and 35 min of ischemia (red tracing/bars) compared to no IR (TC, black tracing/bars) (Fig. 4A); thus RCI was markedly reduced by 2 to 6 fold indicating significant uncoupling of phosphorylation from oxidation (Table 1). In hearts treated during IR with the combined BK_Ca and SK_Ca agonists, NS1619 and DCEB (blue tracing/bars), RCI’s (state 3/state 4) improved under each substrate condition compared to IR alone (Fig. 4).

Table 1.

Modulation of respiration rate in isolated cardiac mitochondria after cardiac IR injury ±BK_Ca + SK_Ca channel agonists.

Pyruvate	state 2	state 3	state 4	RCI
TC	6.7 ±0.7	222.4 ±23.5	11.6 ±3.8	17.6 ±2.6
IR	37.6 ±0.7§	125.3 ±53.2§	69.0 ±17.1§	2.4 ±1.1§
IR+NS1619+DCEB	7.3 ±4.2	221.8 ±31.3*	33.4 ±4.4*§	8.2 ±2.7*§
Succinate	state 2	state 3	state 4	RCI
TC	65.0 ±0.7	457.6 ±38.6	79.7 ±15.4	5.8 ±0.4
IR	73.7 ±9.4	203.7 ±49.0§	114.6 ±7.7§	1.9 ±0.6§
IR+NS1619+DCEB	96.9 ±9.1*§	456.5 ±6.8*	181.5 ±10.3*§	2.8 ±0.2*§
Succinate+rotenone	state 2	state 3	state 4	RCI
TC	63.0 ±10.7	281.4 ±57.0	77.1 ±0.7	3.7 ±0.5
IR	63.0 ±5.4	113.2 ±67.0§	100.5 ±17.7§	1.1 ±0.6§
IR+ NS1619+DCEB	82.7 ±13.7	312.0 ±72*§	138.0 ±4.9§	2.4 ±0.5*§

Respiration rate (O₂ consumption, nmol/min/mg protein) was measured in cardiac mitochondria isolated at 20 min perfusion after 35 min global ischemia ± BK_Ca and SK_Ca channel agonists NS1619 and DCEB given 15 min before and for 20 min after ischemia. RCI (respiratory control index) = state 3/state 4. Time controls (TC) were not subject to ischemia but 75 min perfusion. Note that RCI’s were higher after hearts were treated with NS1619 + DCEB before IR vs. IR alone with pyruvate and succinate + rotenone, but not with succinate. N = hearts/group with 3–4 replicates within each mitochondrial pellet. P<0.05;

^*IR + drugs vs. IR only;

^§IR ± drugs vs. TC.

See also Fig. 3A.

Fig. 4.

Effect of no treatment (TC, no I/R), IR only, or IR + BK_Ca channel agonist NS1619 + SK_Ca channel agonist DCEB, in 12 isolated guinea pig hearts on respiration (A) and Ca²⁺ retention capacity (CRC) (B) in mitochondria isolated after 20 min reperfusion from groups described in Figs. 1,2. The rate of O₂ consumption (respiration) was measured when substrate was added (state 2), followed by ADP (state 3) and after the added ADP was consumed (state 4); substrates added were pyruvate, succinate, or succinate + rotenone (see also Table 1); state 3/state 4 ratio is defined as the respiratory control index (RCI). Uptake of external Ca²⁺ was assessed (Fura-4 fluorescence) in mitochondria energized under the same 3 substrate conditions; increased number of 20 μM CaCl₂ injections (pulses) = increased CRC. Note increased RCI and retained Ca²⁺ (CRC) with combined BK_Ca and SK_Ca agonist treatment vs. IR only. For each group n = 4; RCI and CRC were measured from same mitochondrial pellet from each heart. *^,§P<0.05.

By using the same mitochondrial pellets, we also monitored mitochondrial structural integrity by challenging isolated mitochondria with boluses of 20 μM CaCl₂ added to the respiration buffer (Fig. 4B) until the capability to take up and sequester CaCl₂ (CRC) was exceeded so that mitochondria extrude Ca²⁺ through the so-called mitochondrial permeability transition pore (mPTP). Representative traces of CRC in mitochondria energized under succinate + rotenone conditions are displayed (Fig. 4B, top left) with mean CRC’s under the 3 substrate conditions during state 2 respiration (Fig. 4B); cumulative mitochondrial Ca²⁺ uptake was reduced after IR injury because of early mPTP opening but was greater after hearts were treated with NS1619 and DCEB (Fig. 4B, red and blue tracings). Total average mitochondrial Ca²⁺ uptake before mPTP activation was greater when ex vivo hearts were perfused with NS1619 + DCEB before and after ischemia in mitochondria energized with any of the three substrate conditions (columns Fig. 4B). This indicated that relatively uncoupled mitochondria have elevated mitochondrial Ca²⁺ levels during and after IR as we have shown previously in intact hearts,^{27, 31, 49} or have less capacity to sequester the added CaCl₂. The higher RCIs with BK_Ca and SK_Ca agonist treatment during IR coincided with greater mitochondrial CRCs.

We also determined if BK_Ca and/or SK_Ca antagonism worsened oxidative-phosphorylation assessed by respiration rates and RCI in cardiac mitochondria isolated after 20 min reperfusion and 35 min global ischemia (Table 2, Fig. 5A). The antagonists were given separately and together because we observed different effects of NS8593 when given alone or with PAX on cardiac function and infarct size (Figs. 1–3). Representative traces of O₂ consumption in mitochondria energized with succinate (Fig 5A upper left) are displayed with mean RCI’s under the three substrate conditions (Fig. 5A). Similar to data in Table 1 and Fig. 4A for the agonist experiments, states 2 and 4 respiratory rates were increased, and state 3 respiration was decreased in mitochondria isolated after ischemia and 20 min reperfusion (red tracing/bars) compared to no IR (TC, black tracing/bars) (Table 2, Fig. 5A). Treatment of hearts before and after IR with BK_Ca and or SK_Ca antagonists PAX and NS8593 (green tracing/bar) did not worsen average RCI’s (state 3/state 4) compared to IR alone under any substrate condition, which is different from the cardiac functional and tissue infarction results. This is not unexpected because the respiratory data is derived from an aggregate of functional and dysfunctional mitochondria retrieved during the isolation process and the variability of respiratory measurements may be insufficient to distinguish differences.

Table 2.

Modulation of respiration rate in isolated cardiac mitochondria after cardiac IR injury ±BK_Ca + SK_Ca and/or SK_Ca channel antagonists.

Pyruvate	state 2	state 3	state 4	RCI
TC	6.8 ±0.7	210.2 ±22.4	11.6 ±3.8	18.1 ±2.5
IR	35.7 ±2.3§	112.0 ±33.9§	59.4 ±3.4§	2.2 ±2.0§
IR+PAX	12.0 ±2.6*§	92.3 ±25.2§	53.4 ±3.6§	1.7 ±1.9§
IR+NS8593	30.9 ±2.1*§	145.1 ±37.0§	58.6 ±6.2§	2.3 ±3.0§
IR+PAX+NS8593	20.6 ±1.1*§	96.0 ±20.1§	59.4 ±3.7§	1.6 ±0.8§
Succinate	state 2	state 3	state 4	RCI
TC	64.5 ±0.8	450.4 ±37.5	79.5 ±10.3	5.7 ±0.5
IR	72.0 ±3.2§	220.2 ±32.9§	129.0 ±12.0§	1.7 ±1.2§
IR+PAX	90.1 ±6.1*§	286.2 ±38.8§	154.1 ±14.2§	1.8 ±1.1§
IR+NS8593	168.1 ±8.2*§	280.0 ±53.3§	161.1 ±18.2§	1.7 ±0.8§
IR+PAX+NS8593	116.1 ±9.5*§	246.0 ±38.5§	178.2 ±15.2*§	1.4 ±0.8§
Succinate+rotenone	state 2	state 3	state 4	RCI
TC	62.4 ± 9.8	290.5 ±56.2	76.8 ±0.8	3.8 ±0.6
IR	87.2 ± 6.4	202.0 ±32.9	105.2 ±16.4§	1.9 ±0.7§
IR+PAX	77.4 ±11.6	234.2 ±55.1	136.0 ±33.7§	1.7 ±0.8§
IR+NS8593	96.2 ±14.2§	312.0 ±48.6*	261.3 ±38.2*§	1.2 ±0.8§
IR+PAX+NS8593	108.2 ±26.0§	305.3 ±45.7*	262.3 ±31.6*§	1.2 ±0.7§

Respiration rate (O₂ consumption, nmol/min/mg protein) was measured in cardiac mitochondria isolated at 20 min perfusion after 35 min global ischemia ± BK_Ca and SK_Ca channel antagonists NS8593 ± PAX given 20 min before and for 20 min after ischemia. RCI (respiratory control index) = state 3/state 4. Time controls (TC) were not subject to ischemia but 75 min perfusion. Note that the lower RCI’s after IR were not significantly lower after hearts were treated with PAX ± NS8593 compared to IR alone with any substrate. N = hearts/group with 3–4 replicates within each mitochondrial pellet. P<0.05;

^*IR + drugs vs. IR only;

^§IR ± drugs vs. TC.

See also Fig. 4A.

Fig. 5.

Effect of no treatment (TC, no I/R), IR only, or IR + BK_Ca channel antagonist PAX ± SK_Ca channel antagonist NS8593, in 20 isolated guinea pig hearts on respiration (A) and Ca²⁺ retention capacity (CRC) (B) in mitochondria isolated after 20 min reperfusion from groups described in Figs. 1,2. The rate of O₂ consumption (respiration) was measured when substrate was added (state 2), followed by ADP (state 3) and after the added ADP was consumed (state 4); substrates added were pyruvate, succinate, or succinate + rotenone (see also Table 2); state 3/state 4 ratio is defined as the respiratory control index (RCI). Uptake of external Ca²⁺ was assessed (Fura-4 fluorescence) in mitochondria energized under the same 3 substrate conditions; increased number of 20 μM CaCl₂ injections (pulses) = increased CRC. Note hat the decreased RCI with IR only was not different after treatment with the BK_Ca and or SK_Ca antagonists; in contrast, CRC was reduced after treatment with the BK_Ca and or SK_Ca antagonists in the presence of pyruvate or succinate. For each group n = 4; RCI and CRC were measured from same mitochondrial pellet from each heart. * P<0.05.

Using the same mitochondrial pellets isolated after IR injury, we again monitored mitochondrial structural integrity by challenging isolated mitochondria with boluses of 20 μM CaCl₂ (Fig. 5B). Treatment with the combination of PAX and NS8593 slightly reduced CRC in mitochondria energized with pyruvate or succinate, but not with succinate + rotenone, when compared to IR alone. The reduced CRC after combined BK_Ca and SK_Ca antagonist treatment during IR corroborates the results on cardiac functional and tissue infarction.

Discussion

Our results demonstrate that intrinsic activation of BK_Ca and SK_Ca channels provides endogenous cardiac functional protection (Fig. 1B) in the ex vivo cardiac global IR model on the basis of lowered developed LVP in the presence of PAX alone, and more so in the presence of PAX+NS8593 vs. IR alone; however, only the presence of PAX enhanced global infarct size (Fig. 2B). In contrast, in the in vivo model of regional IR injury (Fig. 3), regional infarct size was larger in the presence of either PAX or NS8593 and even larger with infusion of both PAX and NS8593. The specific agonists for the BK_Ca and the SK_Ca channels, NS1619 and DCEB, provided equivalent protection against IR injury without additive effects. Our protocol for the isolated heart experiments was different from that used in our prior studies in which the BK_Ca and SK_Ca agonists were given only briefly to trigger pharmacologic preconditioning (PPC) and the antagonists were given to prevent PPC.^4,18 The finding that NS8593 alone did not enhance infarct size in the ex vivo global ischemia model as it did in the in vivo LAD occlusion model could be related to many factors, including differences in species, anesthetics given, collateral flow, potential for ischemic or anesthetic preconditioning, methods of inducing ischemia and drug delivery. Nevertheless, the present results suggest that opening or preventing the opening of these K⁺ channels leads to protective or anti-protective effects, respectively, via a common downstream mediator that is likely mitochondrial generated superoxide (O₂^•−) as observed by the loss of protection in the presence of TBAP (Figs. 1E, 2E).

In combination, the BK_Ca and the SK_Ca channel agonists NS1619 and DCEB did not provide additional protection, unlike the combined antagonists that worsened function ex vivo, and enhanced infarct size in vivo. This may indicate that providing either agonist exogenously provides maximal protection, whereas both channels are endogenously important in reducing IR injury. Because the mitochondrial targeted O₂^•− scavenger, TBAP, given before and after ischemia, abrogated the functional recovery and the magnitude of infarction by both agonists alone or in combination, this suggested that the protection was mediated in large part by a mitochondrial effect to stimulate O₂^•− generation. This was further supported not only by effects of BK_Ca and SK_Ca agonists to improve oxidative phosphorylation (RCI) and retain excess mCa²⁺ (CRC) (Fig. 4) but also by effects of BK_Ca and SK_Ca antagonists to reduce Ca²⁺ loading after IR injury (Fig. 5). Overall, our novel observations suggest an important role for IR-induced BK_Ca and SK_Ca channel activation in cardiomyocyte mitochondria in providing endogenous protection against IR injury as suggested by treatment of hearts with either or both channel antagonists.

BK_Ca and SK_Ca Channel Agonists Reduce and Antagonists Enhance Cardiac IR Injury via a Mitochondrial Mechanism

Individually, BK_Ca and SK_Ca channels appear to be important for protecting against cardiac myocyte damage during oxidative stress.^{4, 18, 26, 50–53} Allowing for possible non-specific effects of the agonists and antagonists, our data furnish support that pharmacologic opening of either BK_Ca or SK_Ca channels is cardioprotective. Moreover, intrinsic activation of BK_Ca or SK_Ca channels, and especially when activated together, may be essential for endogenous protection against IR injury. In contrast, because there were no additive effects of the two agonists (Figs. 1A, 2A), protection by either of these agonists may have provided maximal protection via a mK⁺ uptake mechanism.

In our experiments the antagonists were given before, during and after ischemia to test for possible endogenous cardioprotection. Due to different K⁺ channel characteristics and endogenous protective effects we speculate that mSK_Ca channels open gradually under conditions in which ΔΨ_m becomes reduced, pH_m becomes progressively lower, and matrix Ca²⁺ progressively rises, as occurs during ischemia. In contrast, mBK_Ca channel opening may occur when ΔΨ_m rebounds, cytosolic Ca²⁺ surges, and pH_m is high, as during initial reperfusion. This purported timing of opening of these channels, although likely over-simplified, is based on our studies of changes in redox state (NAHD, FAD), diastolic, systolic, and mitochondrial matrix [Ca²⁺], and cytosolic [Na⁺] during the time course of IR injury.^{27, 54–58}

Importantly, the BK_Ca and SK_Ca channel agonists and antagonists were bioactive when infused intravenously, as evidenced by the differences in cardiac infarct size after LAD occlusion in the in vivo rat model (Fig. 3). This suggests a translational value for potential intravenous treatment of K_Ca channel agonists during cardiac IR injury in humans. In fact, we have recently identified splice variants of the SK_Ca channel in human ventricular myocytes,²⁶ which warrants development of SK_Ca-targeted therapeutic strategies for human cardiac IR injury.

The isolated heart and animal studies could not indicate specifically where the anti- and pro-cardioprotective effects of BK_Ca and SK_Ca antagonist and agonists, respectively, occur, i.e. in mitochondria vs. other cellular sites such as the sarcolemma. However, pharmacologic evidence for a role of mitochondrial BK_Ca and SK_Ca channels is given by the improved or worsened RCI’s and CRC’s (Fig. 4A, B vs. Fig. 5A, B) in isolated guinea pig mitochondria after ex vivo IR injury in the presence of SK_Ca and BK_Ca agonists/antagonists. That the protection is mediated at the mitochondrial level is further supported by the effect of the free radical scavenger TBAP to abolish cardioprotection (Figs. 1E, 2E). This is because the respiratory complexes of the mitochondrial electron transport chain are most likely the major source of signaling ROS for cytoprotection and deleterious excess ROS production during IR injury.¹ Indeed, both BK_Ca⁵⁰ and SK_Ca^{18, 26} channels are located in cardiac cell IMM rather than in the sarcolemmal membrane, and drug-induced opening of the BK_Ca channel in isolated mitochondria leads to mild ROS production without significantly affecting ΔΨ_m.⁶ We believe that this small amount of O₂^•− emitted is important for signaling mechanisms that confer protection during treatment with a BK_Ca or SK_Ca agonist.

Distribution and Function of Ca²⁺-Sensitive K⁺ Channels

The cell membranes of vascular smooth muscle, neural, and secretory cells contain large conductance (200–300 pS, i.e. Big Ca²⁺-sensitive K⁺ (BK_Ca, aka maxi-K_Ca) channels that when activated produce vasodilation, hyperpolarization, and secretion in these cells, respectively. BK_Ca channel opening is activated by increased cytosolic [Ca²⁺] and by a large voltage gradient.⁵⁹ Activation of BK_Ca channels over a range of [Ca²⁺] is mediated at several binding sites within the channel⁶⁰ so that there is a wide range of [Ca²⁺] responsiveness (K_a 10–1000 μM).⁶¹ As K⁺ exits the cell with BK_Ca channel opening, this elicits cell membrane repolarization or hyperpolarization, which in turn reduces cell Ca²⁺ entry by closing voltage-dependent Ca²⁺ channels. The finding of altered redox potential in smooth muscle cells⁶² suggested there was a mitochondrial involvement. Indeed, Xu et al.⁵⁰ provided the first evidence that BK_Ca channels are prominently located in cardiac mitochondria. In contrast, SK_Ca channels are not voltage dependent and their agonists interact with their Ca²⁺ sensor calmodulin in the C-terminus region; however, at least for NS8593, the SK_Ca-mediated gating modulation occurs via interaction with gating structures near the selectivity filter within the inner pore vestibule.²⁴

Studies of effects of BK_Ca and SK_Ca channels in cell membranes might help to assess the mechanisms of protection by their counterparts that reside in the IMM. For example, BK_Ca channels are proposed to regulate cell membrane potential in neurons.⁶⁴ SK_Ca channels in neurons lie adjacent to Ca²⁺ stores and Ca²⁺ channels to sense Ca²⁺ activated K⁺ flux.⁶⁵ Neuronal SK_Ca channels appear to play a role in setting the intrinsic firing frequency, whereas neuronal BK_Ca channels may regulate action potential shape.¹⁵ SK_Ca channels are also located in neuronal (hippocampus-derived cells) mitochondria where they appear to provide protection against excitotoxicity.¹⁷

BK_Ca and SK_Ca-mediated K⁺ flux in mitochondria may be differentiated by the number of channels/surface area, sensitivity to Ca²⁺, and dependence on ΔΨ_m during mitochondrial respiration. Because there are several differences in the triggers for activating these channels, the functional effects of opening these channel may be different; e.g., unlike for BK_Ca, SK_Ca channel opening may “fine tune” matrix K⁺ influx due to changing matrix Ca²⁺ levels independent of changes in ΔΨ_m during the variable rate of oxidative phosphorylation. The presence of the SK_Ca channels specifically in the IMM¹⁸ probably indicate that they have an important function in modulating mitochondrial bioenergetics in response to high matrix [Ca²⁺], perhaps via modulation of K⁺-mediated mitochondrial volume that leads to an uncoupling effect⁶³ due to K⁺/H⁺ exchange (KHE) (cartoon, Fig. 6). In contrast, the voltage and Ca²⁺-dependent BK_Ca channels may open primarily when ΔΨ_m and pH_m are restored and excess cytosolic [Ca²⁺] leads to high matrix [Ca²⁺] during reperfusion after ischemia.

Fig. 6.

Schema of proposed interaction of BK_Ca and SK_Ca channels on regulation of mitochondrial K⁺ influx stimulated by increases in cytosolic and mitochondrial [Ca²⁺] and on reciprocal mitochondrial H⁺ influx with K⁺ influx via mitochondrial K⁺/H⁺ exchange (KHE).

Protection by mK⁺ Channel Types Likely Requires Superoxide Release

There is ample evidence that O₂^•− is necessary to trigger protection by a variety of mK⁺ channels;^{1, 3} however, the mechanism of O₂^•−, and its downstream mediators, in mediating protection remain unresolved. An increase in redox state (increased NADH, decreased FAD) at a given [O₂] can result in a small increase in signaling O₂^•− generation.⁶⁶ This is different than the much larger amount of deleterious O₂^•− “bursts” that occur during reperfusion when excess O₂ is available but mitochondrial respiration is compromised. Our group^{4, 28, 29, 67} and others^{68, 69, 70} have shown, moreover, that O₂^•− and its products are also formed in excess during ischemia despite lower tissue O₂ tension.

In the present study, evidence that the protective effects of DCEB and NS1619 are mediated by signaling O₂^•− is again indicated by reversal of the protection in the presence of TBAP as shown previously for SK_Ca^{18, 26} and BK_Ca⁴ channel agonists. Enhanced transfer of electrons by uncoupling before ischemia may minimize respiratory inefficiency; i.e., by reducing matrix contraction and improving respiration on reperfusion. mSK_Ca channel opening, like opening of any mK⁺ channel, could induce protection by a small increase in O₂^•− generation to stimulate enzymatic pathways that help to protect cells from IR injury. In any case, we propose that TBAP accelerates O₂^•− dismutation to H₂O₂ and thus blocks the signaling O₂^•− that leads to protection by either NS1619 or DCEB. We have reported in isolated cardiac mitochondria that low, but not high, concentrations of the BK_Ca channel opener NS1619 increased resting state 4 respiration and mildly enhanced H₂O₂ levels while maintaining ΔΨ_m.⁶

We have suggested, moreover,⁶³ that an increase in intra-matrix K⁺ is replaced over time with H⁺ via mKHE and that at low concentrations, these openers produce a transient increase in matrix acidity, i.e., in which the proton leak stimulates respiration but maintains ΔΨ_m so that signaling O₂^•− is generated at mitochondrial respiratory complexes due to accelerated electron transport. Therefore, during the periods of ischemia and reperfusion, mK⁺ influx, due to condition-dependent activation of SK_Ca and BK_Ca channels, and, in turn, mKHE may trigger a signaling amount of O₂^•−, due to enhanced respiration and electron leak, that then stimulates downstream signaling pathways. The net effect of improved mitochondrial bioenergetics during late ischemia and reperfusion, i.e., higher redox state, lower mCa²⁺, and reduced deleterious O₂^•− emission contributes to improved contractile and relaxant function.^{29, 31, 57, 71}

Ca²⁺-induced mK⁺ Influx Causes Mitochondrial Swelling and Changes in Respiration

We have reported that cytosolic and mCa²⁺ both increase steadily during the period of cardiac ischemia and that cytosolic Ca²⁺ peaks on initial reperfusion.^{27, 49} During IR injury the rise in cytosolic and in mCa²⁺ likely triggers mK⁺ influx via mBK_Ca and mSK_Ca channel activation to initially induce matrix swelling as a potential protective mechanism. Alternatively, or in addition, H⁺ influx via mKHE may enhance respiration to maintain ΔΨ_m but also induce signaling levels of O₂^•− (cartoon, Fig. 6). We observed that PAX, NS1619 and PAX+NS8593 treatments reduced the increase in pyruvate-mediated state two respiration found after IR alone, but increased the succinate-mediated state 2 respiration compared to IR alone (Table 2). This implies that endogenous mK_Ca channel activation does lead to mild uncoupling via mKHE during succinate but not during pyruvate consumption. It is also possible that K⁺ is required for optimal functioning of oxidative phosphorylation because mK⁺ flux largely regulates mitochondrial volume that in turn may modulate bioenergetics.^72–74 Thus mSK_Ca channels, like mBK_Ca channels,^{50, 75} may also act to modulate mitochondrial volume during the increase in matrix Ca²⁺ loading that occurs during ischemia and more so on reperfusion.^{27, 31} Xu et al.⁵⁰ suggested that opening mBK_Ca channels to enhance matrix K⁺ influx mitigates IR injury in a manner similar to putative mK_ATP channel opening. They proposed⁵⁰ that the function of mBK_Ca channels was to improve the efficiency of mitochondrial energy production. In addition to mBK_Ca and mSK_Ca channels, and the putative mK_ATP channels,^{72, 75–79} another K⁺ channel, the Na⁺-activated K⁺ channel, SLO2,^{80, 81} has been reported to be located in mitochondria; this channel is also cytoprotective, at least in part due to activation of mK⁺ influx. Thus, although the mK⁺ channel sensing conditions may be different, the net effect of mK⁺ influx, increases in mitochondrial volume and or mH⁺ via mKHE, appears to be the same.

Because mK_Ca channels likely play a major role in regulating mitochondrial bioenergetics by modulating ion flux, it is unclear exactly how opening of these channels leads to higher NADH/FAD ratios and decreases in excess ROS and in Ca²⁺ overload during IR. For the presumptive mK_ATP channel, it was proposed that its opening depolarizes the IMM and decreases mCa²⁺ overload.^{75, 76, 82} It should be noted that Garlid’s group^{72, 83} has proposed that the physiological role of mK⁺ channel opening is control of mitochondrial volume rather than dissipation of ΔΨ_m and uncoupling, because swelling induced by mK⁺ uptake (without mKHE) would occur with concomitant uptake of Cl⁻ and subsequently water by osmosis. They suggested that subsequent activation of mKHE would only slightly dissipate the proton motive gradient (ΔμH) by increasing matrix acidity (H⁺ leak) without significantly altering ΔΨ_m.^{73, 83}

We found that NS1619 resulted in an increase in mK⁺ uptake, but only in the presence of quinine, a non-specific KHE blocker;⁶³ this suggested that mKHE promoted mK⁺ efflux over time. However, our results also indicated that mpH was lower when quinine was present; this led to the suggestion that mH⁺ influxy induced by NS1619 was not exclusively via mKHE and that NS1619 may also directly or indirectly act as a protonophore. Moreover, valinomycin-induced mK⁺ influx exerted a biphasic effect, i.e. to alkalinize and then to acidify the matrix.⁶³ Thus the mechanism and timing remain unclear. What does appear apparent is that either agonist of mK_Ca channels leads to improved mitochondrial bioenergetics, with reduced mCa²⁺ overload and ROS production, which thereby provides overall substantial mitochondrial and myocyte protective effects during cardiac IR injury ex vivo and reduced infarct size ex vivo and in vivo.

Based on limited experimental observations and some speculation, there are several possible scenarios of potential mechanisms for mBK_Ca and mSK_Ca activation and effectors that lead to cardiac protection (cartoon, Fig. 7). In scenario A, as reperfusion restores cytosolic pH, this promotes excess cytosolic Ca²⁺ loading by sarcolemal NHE and NCE, which then causes excess mCa²⁺ loading with ΔΨ_m repolarization; in turn this activates mK⁺ influx predominately via mBK_Ca but also via mSK_Ca channels due to increased mCa²⁺. In scenario B, as ischemia ensues, an increase in the IMM chemical gradient for Ca²⁺ enhances mCa²⁺ uptake when ΔΨ_m and cell pH are lower; this induces mK⁺ influx predominately via SK_Ca channels but also via BK_Ca channels due to increased cyctosolic Ca²⁺; via either pathway mK⁺ influx is countered by activation of mKHE that acidifies the matrix and promotes uncoupling (higher respiration). In scenario C, independent increases of either cell or mCa²⁺, induced under conditions of ischemia or reperfusion, stimulates mK⁺ influx independently via mBK_Ca and mSK_Ca channels. In each scenario, uncoupling via mKHE leads to reversal of the increase in mitochondrial volume and generation of small amounts of signaling O₂^•−. In each scenario, if the matrix signaling O₂^•− generated by uncoupling were rapidly dismutated to H₂O₂ by TBAP, the protection would be lost because downstream ROS-dependent protective pathways would not be activated. We suggest that scenario B may best explain the utility of independent activation of either type of mK_Ca channel during ischemia and during reperfusion that together protect against cardiac IR injury. This is because we observed that the antagonists together exerted some additive endogenous anti-protective effects that likely depend on the different biochemical conditions that occur during ischemia and during reperfusion.

Fig. 7.

Cartoon summarizes possible scenarios for the interactions of mSK_Ca and mBK_Ca channels and the effects in mediating protection against IR injury. BK_Ca channels might be rapidly activated during reperfusion just after ischemia when ΔΨ_m rapidly becomes fully charged and there is a surge in cytosolic and concomitantly an increase in mitochondrial [Ca²⁺] (A). Independently, SK_Ca channels might be activated more slowly during ischemia when mitochondrial matrix [Ca²⁺] progressively rises in association to the lower ΔΨ_m and pH_m during ischemia (B); in this scenario, BK_Ca and SK_Ca channels might function rather independently to enhance K⁺ influx and trigger K⁺/H⁺ exchange (KHE), cause mild uncoupling, and induce small amounts of signaling ROS. In another alternative pathway, net mK⁺ influx due to SK_Ca channel opening might be affected by simultaneous BK_Ca channel opening to alter either the matrix volume or the degree of mKHE-induced uncoupling (C). In all possibilities, signaling levels of ROS would need to be produced as a result of mK⁺ influx to initiate cardiac protection. A = additive or single pathway, B = parallel pathways with convergence, C = parallel pathways with different mechanisms.

Summary and Limitations

We have furnished comparative evidence for cardiac protective effects of BK_Ca and SK_Ca channel agonists and counter protective effects of BK_Ca and SK_Ca channel antagonists with demonstrated specificity of the antagonists to hinder protection by the agonists. Moreover, systemic application of the agonists and antagonists appears efficacious and suggests therapeutic potential in humans. The end effect of mK⁺ channel opening in cardiac mitochondria appears to be a respiration dependent pathway that stimulates a small amount of signaling mitochondrial O₂^•− that leads to cardioprotection by downstream and local mitochondrial mechanisms as shown by drug treatments on mitochondrial respiration and mCa²⁺ uptake. However, there is as yet little delineation of the individual stages and timing of triggering, activation, and end-effects of mK⁺ channel opening. Unraveling this apparently complicated, and potentially redundant, mechanism that culminates in cardiac protection could lead to therapeutic approaches for protection, specifically via a mitochondrial-targeted mechanism. However, an efficacious therapy would likely require targeting of specific cardiac mitochondrial splice variants of mK_Ca channels, so that the function of other BK_Ca and SK_Ca channels in other organs and subcellular sites would not be impinged.

Summary.

Big and small conductance Ca²⁺ -sensitive K⁺ channel isoforms are present in the inner mitochondrial membrane of cardiac ventricular mitochondria. These channels appear to have a differential protective role against cardiac ischemia reperfusion injury.

Abbreviations

IR

ischemia reperfusion

SK_Ca

small conductance Ca²⁺ -sensitive K⁺ channel

BK_Ca

big conductance Ca²⁺ -sensitive K⁺ channel

FAD

flavin adenine dinucleotide

KHE

K⁺/H⁺ exchange

NCE

Na⁺/Ca²⁺ exchange

NHE

N⁺/H⁺ exchange

K_ATP

ATP -sensitive K⁺ channel

DCEBIO

DCEB, 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one

NADH

nicotinamide adenine dinucleotide

NS1619

(1-(2′-hydroxy-5′-trifluoromethylphenyl)-5-trifluoromethyl-2(3H) benzimid-axolone)

IMM

inner mitochondrial membrane

ΔΨ_m

mitochondrial membrane potential

TBAP

Mn(III) tetrakis (4-benzoic acid) porphyrin

IPC

ischemic preconditioning

PPC

pharmacological preconditioning

PAX

paxilline

NS8593

N-[(1R)-1,2,3,4-tetrahydro-1-naphthalenyl]-1H-benzimidazol-2-amine hydrochloride

RT-PCR

reverse transcription -polymerase chain reaction

RCI

respiratory control index

TTC

2,3,5-triphenyltetrazolium chloride

CRC

Ca²⁺ retention capacity

CaM

calmodulin