The activation of ATP-sensitive potassium (KATP) ion channels in the heart is thought to exert a cardioprotective effect under low oxygen conditions, possibly enhancing tolerance of environmental hypoxia in aquatic vertebrates. The purpose of this study was to examine the possibility that hypoxia-induced activation of cardiac KATP channels, whether in the sarcolemma (sarcKATP) or mitochondria (mitoKATP),enhances viability in cardiac muscle cells from a species highly tolerant of low oxygen environments, the goldfish Carassius auratus. During moderate hypoxia (6–7 kPa), the activation of sarcKATPchannels was indicated by a reduction in transmembrane action potential duration (APD). This response to hypoxia was mimicked by the NO-donor SNAP(100 μmol l–1) and the stable cGMP analog 8-Br-cGMP, but abolished by glibenclamide or l-NAME, an inhibitor of NO synthesis. The mitoKATP channel opener diazoxide did not affect APD. Isolated ventricular muscle cells were then incubated under normoxic and hypoxic conditions. Cell viability was decreased in hypoxia; however, the negative effects of low oxygen were reduced during simultaneous exposure to SNAP,8-Br-cGMP, and diazoxide. The cardioprotective effect of diazoxide, but not 8-Br-cGMP, was reduced by the mitoKATP channel blocker 5-HD. These data suggest that hypoxia-induced activation of sarcKATP or mitoKATP channels could enhance tolerance of low-oxygen environments in this species, and that sarcKATP activity is increased through a NO and cGMP-dependent pathway.
Many studies suggest that the activation of cardiac muscle ATP-sensitive potassium (KATP) channels can play a beneficial role during hypoxia and ischemia in mammals (Gross and Peart,2003; Grover and Garlid,2000; O'Rourke,2000). This conclusion is in accord with the original hypothesis of Noma (1983), who was the first to characterize sarcolemmal KATP (sarcKATP)channels. These are activated when intracellular oxygen levels and ATP/ADP ratios are low, although the precise function of sarcKATP channels in cardiac muscle is unknown (Flagg et al.,2004). Since the subsequent discovery that KATPchannels are also located in the mitochondrial inner membrane(mitoKATP; Inoue et al.,1991), there has been considerable debate as to the relative roles played by sarcKATPvs mitoKATP channels in cardioprotection and ischemic preconditioning(Gross and Peart, 2003; Toyoda et al., 2000). KATP channel activation under depleted oxygen conditions is often linked to the prior production of nitric oxide (NO), which acts through a cGMP-dependent mechanism (Dawn and Bolli,2002; Wang et al.,2001).
The observation that hypoxia-induced KATP channel activation is beneficial in mammals raises the possibility that a similar response in aquatic ectotherms could promote tolerance of environmental hypoxia. SarcKATP channel currents have been recorded in isolated myocytes from the hearts of several teleost species(Ganim et al., 1998; Paajanen and Vornanen, 2002),and there is evidence for the existence of cardiac mitoKATPchannels in fish (MacCormack and Driedzic,2002). In addition, nitric oxide synthase (NOS) has been localized in the hearts of goldfish (Bruning et al.,1996), and NO has been shown to play a critical role, viacGMP, in regulating the heart and peripheral vasculature in a variety of teleost species (Imbrogno et al.,2003; Jennings et al.,2004; Pellegrino et al.,2003). In goldfish, NO synthesized in cardiac myocytes plays a role in sarcKATP channel activation during moderate hypoxia(Cameron et al., 2003). Although it has been proposed that hypoxia-induced KATP channel activation, whether in the heart or in the brain, contributes to the enhanced tolerance of environmental oxygen depletion exhibited by many ectothermic vertebrates (Cameron and Baghdady,1994; Pek-Scott and Lutz,1998), this possibility has not been directly tested in fish.
The purpose of the present study, then, was to determine whether sarcKATP or mitoKATP channel activation exerts a cardioprotective effect during hypoxia in goldfish. This species is highly tolerant of depleted environmental oxygen; indeed, the congeneric crucian carp Carassius carassius retains normal cardiac function for 5 days of complete anoxia (Stecyk et al.,2004). A cellular model of environmental hypoxia was used to examine the effects of altered channel activity on isolated myocytes. Our hypothesis was that the NO- and cGMP-dependent activation of cardiac KATP channels would promote survival of isolated cells, thereby contributing to a suite of mechanisms that dramatically enhance tolerance of hypoxia in these fish. Preliminary results of this work have appeared in abstract form (Zhu et al.,2004).
Materials and methods
Goldfish Carassius auratus L. were obtained from local suppliers and acclimated at 21°C in filtered, aerated 200 liter aquaria under a 12 h:12 h light:dark cycle. On the day of an experiment, goldfish of either sex were anesthetized by immersion in a solution of MS-222 (tricaine methanesulfonate, 0.2 g l–1; pH 7.6). The heart was rapidly excised and prepared for standard intracellular recording using the intact ventricle, or for enzymatic dispersion of individual myocytes.
Animals used for conventional intracellular recording ranged in size from 9.5–28.1 g (mean ± s.e.m.,18.1±4.8 g). Procedures have been previously described(Cameron et al., 2003);briefly, the entire heart was mounted in a tissue bath and superfused with normoxic saline solution at a rate of 15 ml min–1. This solution contained (in mmol l–1) 150 NaCl, 5 KCl, 1.2 NaH2PO4, 1.2 MgSO4, 1.8 CaCl2, 10 Hepes and 10 glucose; pH was maintained at 7.6 and temperature held at 21±1°C.
Standard glass microelectrodes filled with 3 mol l–1 KCl were connected to a differential amplifier (WPI, Sarasota, FL, USA), and spontaneous intracellular action potentials (APs) were recorded from the most superficial layer of ventricular muscle. APs were digitized (PowerLab;ADInstruments, Mountain View, CA USA) and analyzed using appropriate software(Chart, Peak Parameters; ADI). Parameters measured were action potential amplitude, resting membrane potential, slope of action potential upstroke phase and action potential duration at various levels of repolarization.
Oxygen partial pressure in the tissue bath was monitored using an oxygen meter (Cameron Instruments, Port Aransas, TX, USA) while moderate hypoxia(6.1±0.2 kPa) was induced by switching the superfusate from air-bubbled saline (20 kPa) to a glucose-free solution gassed with 100% N2. The new condition was reproducible, reversible, and achieved in the tissue bath within 1 min. Electrophysiological responses to the acute onset of hypoxia and to reoxygenation were first assessed in the absence of any drug. Recordings were collected after 10–30 min exposure to hypoxia, and again 20 min after a return to normoxic solution.
Drugs, including the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP; 100 μmol l–1), the nitric oxide synthase (NOS) inhibitor Nω-Nitro-l-arginine methyl ester (l-NAME;50 μmol l–1), the KATP channel antagonist glibenclamide (5 μmol l–1), the mitoKATPchannel agonist 3-methyl-7-chloro-1,2,4-benzothiadiazine-1,1-dioxide(diazoxide; 100 μmol l–1), the stable and cell-permeable cGMP analog 8-bromoguanosine 3′:5′-cyclic monophosphate(8-Br-cGMP; 100 μmol l–1), and the mitoKATPchannel inhibitor 5-hydroxydecanoic acid (5-HD; 100 μmol l–1), were purchased from Sigma (St Louis, MO, USA). Diazoxide was first dissolved in a drop of DMSO. Following the initial period of equilibration in normoxic saline, the effects of SNAP, 8-Br-cGMP and diazoxide on ventricular action potential parameters were recorded after 10 min exposure. In other experiments, glibenclamide or l-NAME was made up in hypoxic solution and applied to the tissue bath as described above. Dosages were chosen from among those recently employed in comparable studies.
Cellular model of environmental hypoxia
Isolation of cardiac myocytes
Animals used for this procedure ranged in size from 23.7 to 64.9 g (mean± s.e.m., 43.3±12.4 g). Cell isolation procedures were modified from those of Karttunen and Tirri(1986), as previously described (Cameron et al.,2003). Upon removal, the heart was bathed in a chilled Ca2+-containing saline buffer (μmol l–1): 137 NaCl, 4.6 KCl, 3.5 NaH2PO4, 1.2 MgCl2, 11 glucose, 10 Hepes and 0.025 CaCl2. The atrium was tied off and an olive-tipped cannula (27 gauge) was inserted into the ventricle through the opening of the bulbus arteriosus. The ventricle was then perfused with an identical but nominally Ca2+-free saline solution for 5 min,followed by 25 minperfusion with an enzyme buffer containing 50 ml Ca2+-free saline, 35 mg collagenase (Type I), 25 mg trypsin and 25 mg bovine serum albumin (Fraction V). The heart was removed from the perfusion apparatus and carefully torn into small pieces. After 5 min of swirling agitation followed by 5 min of gentle trituration with a Pasteur pipette, the supernatant containing single free-floating cells was removed. Fresh buffer was added, and the procedure repeated to obtain a second and third fraction of cells. This procedure produced a high yield (80–90%) of viable,elongated myocytes.
The method used to assess cellular viability in hypoxia was derived from the in vitro model of cellular ischemia developed by Vander Heide et al. (1990), as modified by Sato et al. (2000). Portions of the myocyte suspension (0.5 ml) were pipetted into 5 ml test tubes, and 0.5 ml of saline buffer or drug solution was added. The hypoxic condition was induced in some tubes by layering an additional 0.5 ml of mineral oil on top of the myocyte suspension, preventing gaseous diffusion of oxygen. After 60 min incubation, 0.5 ml of myocytes were sampled from each tube and mixed with 0.5 ml of staining solution containing 0.5% gluteraldehyde and 0.5% Trypan Blue in saline buffer. The resulting ml of suspended cells was placed on a slide and examined by phase contrast microscopy at 100×. Elongated myocytes with a normal morphological appearance were included in the analysis,while dead, `rounded-up' cells were excluded(Vander Heide et al., 1990). These dead cells were always blue, and were presumed to have been damaged during the enzymatic isolation procedure. Viability of the elongated cells was assessed by their capacity to exclude Trypan Blue(Fig. 1); the percentage staining clearly after 3 min exposure to the staining solution was recorded for each experimental condition. Myocytes staining dark blue were considered irreversibly damaged. Drugs and concentrations were identical to those used in the intracellular experiments; they were made up in saline buffer on the day of an experiment and were present throughout the period of in vitrohypoxia.
Data obtained from the intracellular and cellular hypoxia experiments were transferred to Microsoft Excel spreadsheets; t-tests and analysis of variance for repeated measures (ANOVA with post-hoc Fisher PLSD and Scheffe F-test; Statview) were used to determine statistical significance (P<0.05) among group means; results were expressed as means ± s.e.m.
Effects of hypoxia
Transmembrane intracellular recording techniques were used to monitor the effects of moderate hypoxia (6.1±0.2 kPa) on the configuration of ventricular action potentials in goldfish heart. Cardiac action potential duration (APD) was decreased in all experiments (N=5). As previously observed (Cameron et al.,2003), there was a significant (P<0.01) reduction in APD at 90% of full repolarization (APD90) after 10 min of exposure to substrate-free hypoxia (Fig. 2). Mean APD90 under normoxic conditions was 575±24 ms; after exposure to hypoxia, this value fell to 488±12 ms, a decrease of 15.1%. APD at 50% of full repolarization (APD50)was not significantly reduced. There were no significant effects of hypoxia on any other electrophysiological parameters, including resting membrane potential, action potential amplitude or slope of action potential upstroke.
Glibenclamide and l-NAME in hypoxia
To examine the possibility that APD shortening in hypoxia was caused by the activation of KATP channels, the effects of the channel antagonist glibenclamide (5 μmol l–1) on hypoxia-induced shortening were monitored in isolated hearts. Simultaneous exposure to both glibenclamide and hypoxia abolished the reduction in APD characteristic of hypoxia alone(N=3; Fig. 3).
To determine whether NO plays a role in cellular responses to hypoxia in goldfish, the effects of the NOS inhibitor l-NAME were studied in isolated hearts and in ventricular myocytes. Hypoxia-induced shortening of APD90 was eliminated by previous exposure to 50 μmol l–1l-NAME (N=4; Fig. 3).
Effects of KATP activators under normoxic conditions
To determine whether NO and cGMP could mimic cellular responses to hypoxia,the effects on action potential parameters of the NO donor SNAP and the stable cGMP analog 8-Br-cGMP were monitored under normoxic conditions. At a concentration of 100 μmol l–1, SNAP significantly(P<0.05) reduced APD90 after 10 min exposure(N=4; Fig. 4). With prolonged exposure to SNAP, APD was further reduced; at 60 min, both APD50 and APD90 were significantly decreased. There were no significant effects of SNAP on any other action potential parameter.
Cellular viability in vitro
A cellular model of environmental hypoxia was used to assess the potential cardioprotective effects of KATP channel activation. Under normoxic conditions, 37.8±4.0% of elongated, isolated ventricular myocytes from goldfish were unable to exclude Trypan Blue, suggesting cellular injury(N=6; Fig. 5). After 60 min hypoxia, however, the incidence of Trypan Blue-positive cells was significantly (P<0.001) increased compared to that observed in normoxia. The percentage of stained cells in hypoxia was 81.2±2.6, a 115% increase. More than 500 myocytes were individually evaluated.
Following simultaneous exposure of isolated myocytes to l-NAME and 60 min hypoxia, the percentage of Trypan Blue-stained cells was moderately increased relative to that seen with hypoxia alone(Fig. 6). After 60 min exposure of isolated myocytes to hypoxia plus SNAP, however, the percentage of Trypan Blue-stained cells was markedly reduced compared to that observed with hypoxia alone. To determine whether this cardioprotective action of SNAP was linked to the hypoxia-induced production of NO in muscle cells, the effect of SNAP in combination with l-NAME also was tested. Blockade of NO synthesis by l-NAME entirely eliminated the reduction in stained cells observed with SNAP and hypoxia alone (Fig. 6).
Selective activation of sarcKATP vs mitoKATP channels
In isolated cells exposed to hypoxia, diazoxide exerted a marked cardioprotective influence. Isolated myocytes exposed to diazoxide during 60 min hypoxia survived in greater numbers than did those affected by hypoxia alone (Fig. 7).
In mammals, diazoxide and 5-HD have been shown to be selective for mitoKATP over sarcKATP channels — the former as an activator and the latter as an inhibitor(Mannhold, 2004). In goldfish,the beneficial effects of diazoxide in preserving isolated myocytes was reduced, but not eliminated, by 5-HD (Fig. 7). In contrast, 5-HD did not alter the cardioprotective effect of 8-Br-cGMP; the reduction in the percentage of stained cells in hypoxia that was produced by 8-Br-cGMP alone was not affected when this agent was given in combination with the presumed mitoKATP blocker.
The present findings support the hypothesis that hypoxia-induced activation of cardiac sarcolemmal or mitochondrial KATP channels in goldfish protects muscle cells during metabolic stress, possibly enhancing whole-animal tolerance of environmental hypoxia. The cardioprotective activation of KATP channels in this species, whether in whole hearts or isolated myocytes, is linked to the production of nitric oxide, which acts through a cGMP-dependent mechanism.
In the present study we confirm our previous observation(Cameron et al., 2003) that transmembrane action potential duration is significantly shortened during moderate hypoxia in intact, isolated goldfish heart(Fig. 2). In mammals, cardiac APD shortening in hypoxia is thought to arise through the activation of sarcKATP channels (Flagg et al.,2004; Shigematsu and Arita,1997; Suzuki et al.,2002). Patch clamp studies also have revealed a hypoxia-induced increase in the activity of sarcKATP channels in both mammals(Budas et al., 2004; Liu et al., 2001) and goldfish(Cameron et al., 2003). We now show that in fish, the APD shortening can be eliminated by simultaneous exposure of the tissue glibenclamide (Fig. 3), an antagonist of both sarc- and mitoKATP channels. The hypoxia-induced reduction in APD was also abolished by l-NAME,an inhibitor of NO synthase, suggesting an upstream role for NO in the activation of sarcKATP channels(Fig. 3). Further, APD shortening can be similarly induced by the NO donor SNAP, as well as by a stable analog of cGMP (Fig. 4). NO has previously been shown to activate sarcKATP channels in mammalian cardiac muscle (Chen et al.,2000; Moncada et al.,2000), and this response has been linked to cGMP(Chen et al., 2000; Han et al., 2001). In contrast, the mitoKATP activator diazoxide had no significant effect on APD.
Cardioprotection in vitro
In an attempt to demonstrate that hypoxia-induced and NO-dependent activation of KATP channels promotes the survival of ventricular myocytes, we used an in vitro model of environmental hypoxia. Similar methods have been used to simulate the effects of ischemia on isolated mammalian cells, and to demonstrate, for example, the cardioprotective activation of mitoKATP channels by diazoxide(Sato et al., 2000) or volatile anesthetics (Zaugg et al.,2002). In each study, the viability of apparently intact,elongated myocytes was assessed by their capacity to exclude Trypan Blue dye.
The present results indicate that ventricular myocytes from goldfish are damaged after 60 min of in vitro hypoxia under mineral oil. As illustrated in Fig. 5, cells exposed to the low-oxygen environment were significantly more often positive for Trypan Blue than those maintained under normoxic conditions. However, the cellular damage caused by hypoxia could be reduced by agents thought to activate KATP channels. Cells exposed to hypoxia in the presence of SNAP or 8-Br-cGMP, both of which reduced APD in the intact heart, were much more likely to survive and exclude the dye than were cells exposed to hypoxia alone (Figs 6, 7). In contrast, the NOS inhibitor l-NAME, which prevented hypoxia-induced APD shortening,increased the numbers of stained, damaged cells. Perhaps surprisingly, l-NAME also eliminated the cardioprotective influence of SNAP,suggesting that SNAP promotes the endogenous synthesis and release of NO from cardiac muscle cells. Confounding this analysis is the fact that hypoxia itself may increase the NO concentration in cardiac muscle through activation of NOS isoforms (which vary from one species to the next), or through NOS-independent pathways (Schulz et al.,2004). SNAP has been shown to increase the concentration of cGMP in mammalian myocardium (Qin et al.,2004).
The present data strongly suggest that NO, in its capacity to activate sarcKATP channels in goldfish, promotes survival of cardiac muscle cells during hypoxia. The cardioprotective, cGMP- and KATP-dependent influence of NO in hypoxia has been recently reviewed (Kolar and Ostadal,2004; Schulz et al.,2004). In mammalian myocytes, NOS is upregulated during ischemia(Wang et al., 2002), and a similar enhancement of NOS activity occurs in response to hypoxia in the vasculature of an elasmobranch fish(Renshaw and Dyson, 1999).
SarcKATP vs mitoKATP
While a cardioprotective role for KATP channel activation has been described by many investigators, there remains some question as to the relative roles of sarcolemmal vs mitochondrial channels(Gross and Peart, 2003). In the present study, the cellular damage induced by hypoxia could be reduced by agents thought to specifically activate either sarcKATP or mitoKATP channels. SNAP and 8-Br-cGMP, which reduce APD through activation of sarcKATP channels, also promote tolerance of hypoxia in isolated cells (Figs 6, 7). Diazoxide, which is thought to act primarily at mitoKATP, also decreased the numbers of stained, damaged myocytes (Fig. 7). In the present study, diazoxide did not affect APD(Fig. 4), supporting the view that its effect is primarily at the mitochondrial channel. However, a recent study involving rat ventricular myocytes during metabolic inhibition indicates that this agent can open sarcKATP channels as well, probably by an indirect mechanism (Rodrigo et al.,2004). Finally, 5-HD, a specific inhibitor of mitoKATPin mammals, was found to reduce (but not eliminate) the cardioprotective influence of diazoxide, but to have no effect on the beneficial response to 8-Br-cGMP.
Data arising from mammalian studies of hypoxia and ischemic preconditioning also suggest that activation of either sarcKATP or mitoKATP channels can be cardioprotective(Sanada et al., 2001; Tanno et al., 2001; Toyoda et al., 2000). Studies of transgenic mice expressing a mutant KATP channel with reduced ATP-sensitivity suggest that the sarcolemmal channel, at least in mammals, is required for optimal response to ischemia(Rajashree et al., 2002). In pharmacological studies, sarcKATP activation protects against ischemic injury (Suzuki et al.,2002), and the present data suggest a similar action against hypoxia in fish (Fig. 6). In contrast, Sato et al. used an in vitro model of cellular ischemia,very similar to that employed in the present study, to show that activation of mitoKATP channels, and not sarcKATP channels, was responsible for ischemic cardioprotection in rabbit myocytes(Sato et al., 2000). Indeed,many recent studies, most involving diazoxide, 5-HD and other agents presumed to be selective for cardiac sarcKATPvsmitoKATP channels, implicate the latter subtype in protection against ischemia (Murata et al.,2001; Oldenburg et al.,2002; Uchiyama et al.,2003).
There has been considerable speculation as to the specific benefits provided to the myocardium by sarcKATP or mitoKATPchannel activation (Gross and Fryer,1999; Oldenburg et al.,2002). Recent evidence suggests that NO/cGMP-dependent cardioprotection involves mitoKATP channels, not as an end-effector, but as an element in a cascade of events leading to the production of reactive oxygen species (ROS; Lebuffe et al., 2003; Qin et al., 2004; Xu et al., 2004).
One factor that hinders the interpretation of data in studies such as these is that many of the pharmacological agents typically used, including several of those employed in the present study, may protect the heart by means unrelated to ion channel activation. It has been reported that the cardioprotective influence of SNAP, for example, may involve the mitochondria and be unrelated to its capacity to affect sarcKATP channels(Rakhit et al., 2001). Both diazoxide and 5-HD can have beneficial but channel-independent effects on mitochondrial metabolism (Dzeja et al.,2003; Hanley et al.,2002), leading at least one group to challenge the existence of diazoxide- and 5-HD-sensitive KATP channels in mammalian mitochondria (Das et al.,2003). In addition, preliminary data from our laboratory suggest that 5-HD eliminates the reduction in APD characteristic of hypoxia,suggesting that this agent is not specific to mitoKATP channels in fish, or that other channels are affected. The present data cannot exclude the possibility that diazoxide benefits goldfish myocytes by means other than mitoKATP channel activation. This could underlie our observation that 5-HD reduced, but did not completely eliminate, the cardioprotective effects of diazoxide (Fig. 7).
It is now widely accepted that the NO- and cGMP-dependent activation of sarcKATP and/or mitoKATP channels is cardioprotective under conditions of metabolic stress in mammals, and that channel activity underlies the phenomenon of ischemic preconditioning(Gross and Peart, 2003; Kolar and Ostadal, 2004). Aquatic ectotherms may be regularly exposed to acute or chronic environmental hypoxia. Moreover, both sarcKATP(Cameron et al., 2003; Paajanen and Vornanen, 2002)and mitoKATP (MacCormack and Driedzic, 2002; MacCormack et al., 2003) channels exist in the hearts of teleost fish, and the former are specifically activated in hypoxia by a NO-dependent mechanism(Cameron et al., 2003). The present findings support the hypothesis that sarcKATP or mitoKATP channel activation promotes the survival of ventricular myocytes exposed to conditions of depleted oxygen in vitro. A similar channel activation in critical tissues in vivo may extend whole-animal tolerance of hypoxia.
The authors gratefully acknowledge the assistance of Lilian G. Perez and Natasha K. Soodoo in conducting some experiments. This project was funded in part by a grant from the National Science Foundation (DBI-0097499) and by Brachman Hoffman and other grants from Wellesley College.