| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
2 Department of Pediatrics and the Cardiovascular Research Center, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908; and 1 The Rotary Centre for Cardiovascular Research, Griffith University Gold Coast Campus, Southport, QLD 4217 Australia
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
We studied the role of mitochondrial ATP-sensitive K+ (KATP) channels in modifying functional responses to 20 min global ischemia and 30 min reperfusion in wild-type mouse hearts and in hearts with ~250-fold overexpression of functionally coupled A1-adenosine receptors (A1ARs). In wild-type hearts, time to onset of contracture (TOC) was 303 ± 24 s, with a peak contracture of 89 ± 5 mmHg. Diastolic pressure remained elevated at 52 ± 6 mmHg after reperfusion, and developed pressure recovered to 40 ± 6% of preischemia. A1AR overexpression markedly prolonged TOC to 517 ± 84 s, reduced contracture to 64 ± 6 mmHg, and improved recovery of diastolic (to 9 ± 4 mmHg) and developed pressure (to 82 ± 8%). 5-Hydroxydecanoate (5-HD; 100 µM), a mitochondrial KATP blocker, did not alter ischemic contracture in wild-type hearts, but increased diastolic pressure to 69 ± 8 mmHg and reduced developed pressure to 10 ± 5% during reperfusion. In transgenic hearts, 5-HD reduced TOC to 348 ± 18 s, increased postischemic contracture to 53 ± 4 mmHg, and reduced recovery of developed pressure to 22 ± 4%. In summary, these data are the first to demonstrate that endogenous activation of KATP channels improves tolerance to ischemia-reperfusion in murine myocardium. This functional protection occurs without modification of ischemic contracture. The data also support a role for mitochondrial KATP channel activation in the pronounced cardioprotection afforded by overexpression of myocardial A1ARs.
contracture; ischemia; mouse; reperfusion; transgenic
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
A RANGE OF ENDOGENOUS CARDIOPROTECTIVE mechanisms exists within the heart, providing some tolerance to ischemia-reperfusion. These include (but are not restricted to) heat shock proteins; antioxidant enzymes (glutathione peroxidase, superoxide dismutase, catalase); receptor systems, including A1- and A3-adenosine receptors; and KATP channels. The importance of these mechanisms in determining intrinsic tolerance to ischemia (i.e., in the absence of exogenous activation) is unclear. The potential role of ATP-sensitive K+ (KATP) channels during ischemia-reperfusion has received considerable attention in recent years. Exogenous activation of KATP channels provides functional and metabolic benefit during ischemia and reperfusion (7, 11, 13, 14, 17, 18, 29, 30), and KATP channel blockade reduces beneficial effects of preconditioning (1-4, 18, 21, 25, 30, 37, 41) and adenosine receptor stimulation (18, 25, 38-41). It has become evident that this myocardial protection may be mediated via mitochondrial rather than sarcolemmal KATP channels (4, 12, 14, 22, 26). Despite an abundance of evidence supporting protection by exogenous activation, the role of intrinsically activated KATP channels in determining tolerance to ischemic insult remains controversial. Similarly, the potential involvement of KATP channels in A1-adenosine receptor (A1AR)-mediated protection is also unclear. The purpose of the present study was twofold: 1) to test the hypothesis that intrinsic activation of mitochondrial KATP channels does enhance myocardial tolerance to ischemia-reperfusion and 2) to test the hypothesis that KATP channel activation contributes to the marked cardioprotection afforded by transgenically overexpressed A1ARs in murine myocardium (19, 28, 32). To achieve these goals, we examined the functional effects of the selective mitochondrial KATP blocker 5-hydroxydecanoate (5-HD) (3, 17, 21, 25, 26, 29, 37, 40, 41) during global normothermic ischemia and reperfusion in isovolumically functioning hearts from wild-type mice and from mice transgenically overexpressing cardiac A1ARs (19, 28).
| |
MATERIALS AND METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Langendorff perfused murine heart model. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals [DHEW Publication No. (NIH) 85-23, Revised 1996, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20205], and the work was approved by the institutional animal experimental ethics committees. Transgenic mice overexpressing myocardial A1ARs were produced as described in detail recently (19, 28, 32). Hearts were isolated from 7- to 12-wk-old male and female wild-type (23.35 ± 1.12 g body wt, 123 ± 9 mg wet heart wt, n = 26) and transgenic mice (25.02 ± 1.10 g body wt, 137 ± 10 mg wet heart wt, n = 24). Specifically, mice were anesthetized with 50 mg/kg pentobarbital sodium administered intraperitoneally, a thoracotomy was performed, and hearts were rapidly excised into heparinized ice-cold perfusion fluid. The aorta was cannulated (20-gauge stainless steel tube), and the hearts were retrogradely perfused at a pressure of 80 mmHg with modified Krebs buffer containing (in mM): 118 NaCl, 25 NaHCO3, 4.7 KCl, 1.2 KH2PO4, 2.5 CaCl2, 1.2 Mg2SO4, 11 glucose, and 0.5 EDTA. Buffer was equilibrated with 95% O2-5% CO2 at 37°C, giving a pH of 7.4. The left ventricle was vented with a small polyethylene apical drain, and a fluid-filled balloon constructed of plastic film was inserted into the left ventricle via the mitral valve. The balloon was connected to a pressure transducer by polyethylene tube to permit continuous measurement of left ventricular pressure. Hearts were immersed in perfusate maintained at 37°C and the ventricular balloon was inflated to yield a left ventricular diastolic pressure of 2-4 mmHg. Coronary flow rate was continuously monitored via a Doppler flow probe (Transonic Systems, Ithaca, NY) in the aortic perfusion line. Functional data were recorded on a MacLab data acquisition system (ADInstruments, Castle Hill, Australia) connected to an Apple 7300/180 computer. The ventricular pressure signal was digitally processed online (using MacLab Chart 3.5.6, ADInstruments) to yield heart rate and ±dP/dt. All hearts were stabilized for 30 min before experimentation.
A1AR density analysis.
Hearts from transgenic positive and negative (wild-type mice) animals
were homogenized in 10 vol of ice-cold buffer (in mM: 10 EDTA, 10 HEPES, 0.1 benzamidine, pH 7.4). The homogenate was centrifuged at
48,000 g for 10 min, and the pellet was resuspended in 30 ml
of buffer with EDTA reduced to 1 mM. This was centrifuged and
resuspended twice, with the final pellet resuspended in 1 vol of
buffer. Membrane suspensions were stored at 
80°C until analyzed for
binding. A1AR density in cardiac membranes was determined by quantitation of specific binding of the A1 receptor
antagonist, 8 cyclopentyl-1,3-[3H]dipropylxanthine
(DPCPX, 0.1-7.5 nM), and the dissociation constant (Kd) was determined using standard techniques as
described in detail previously (28). Protein content was
determined by the Lowry method using bovine serum albumin as standard.
Experimental protocol. After 20 min stabilization at intrinsic heart rate, all hearts (wild-type and transgenic) were paced at a constant rate of 395 ± 10 beats/min (ventricular pacing with 5-ms square waves 20% above threshold, typically 2-3 V). After a further 10-min period, baseline measurements were recorded and 20 min global normothermic ischemia was initiated by clamping the aortic cannula. Ventricular pacing was stopped on initiation of ischemia. Time to onset of contracture during global ischemia was calculated as the time from cessation of coronary perfusion to the point when diastolic pressure rose by 20 mmHg. Additionally, the maximal left ventricular contracture pressure achieved during ischemia was measured in each heart. After ischemia, hearts were reperfused for 30 min with ventricular pacing resumed after 2 min of reperfusion. Studies were performed in untreated hearts (n = 8 for wild-type, n = 7 for transgenic) or hearts treated with 100 µM 5-HD before ischemia (n = 7 for wild-type, n = 9 for transgenic). 5-HD (Research Biochemicals, Natick, MA) was dissolved directly in perfusion fluid. The infusion was initiated 10 min before ischemia and was maintained for the duration of the experiment (stopped during the ischemic insult). A subset of 5-HD-treated wild-type hearts (n = 5) were manually perfused at the coronary flow rate observed in untreated wild-type hearts using a Gilson eight-roller peristaltic pump. This was done to assess the relative contribution of the inhibition of hyperemia to impaired functional recovery in 5-HD-treated hearts.
Statistical analysis. All results are expressed as means ± SE. Functional parameters under baseline conditions and during ischemia-reperfusion were statistically analyzed by a two-way analysis of variance for repeated measures with Tukey's honestly significant difference post hoc test. In all tests, significance was accepted for P < 0.05.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
A1AR properties in membranes from wild-type and transgenic hearts. Radioligand binding revealed expression of 8 ± 1 fmol/mg protein of A1ARs in membrane suspensions from wild-type hearts. In transgenic hearts, a total of 2,177 ± 375 fmol/mg protein was present. Thus degree of A1AR overexpression is ~250-fold. The Kd values for DPCPX binding were calculated to be 0.7 ± 0.2 nM in wild-type hearts and 1.1 ± 0.1 nM in transgenic hearts. These values are in agreement with Kd values determined for DPCPX at A1ARs in other studies.
Functional responses in wild-type vs. transgenic hearts.
Baseline functional parameters for hearts from wild-type and transgenic
animals (before pacing) are shown in Table
1. Intrinsic heart rate was slightly
higher in wild-type vs. transgenic hearts. Preischemic contractile
function was similar in both groups (Table 1). Global normothermic
ischemia completely abolished contractile function in all hearts within
2-4 min and caused a rapid rise in diastolic pressure
(contracture). Ischemic injury assessed by time to contracture was much
greater in wild-type vs. transgenic hearts. Time to onset of
contracture was ~300 s in wild-type and ~520 s in transgenic hearts
(Fig. 1A). The maximal degree
of ischemic contracture achieved was ~90 mmHg in wild-type hearts vs.
only 60 mmHg in transgenic hearts (Fig. 1B). During 30 min
of reperfusion, there was a sustained depression of left ventricular
developed pressure and an elevation in diastolic pressure in all groups (Fig. 2). However, whereas diastolic
pressure recovered minimally to 52 ± 6 mmHg in wild-type hearts,
it recovered to below 9 ± 4 mmHg in transgenic hearts and did not
differ significantly from preischemia (Fig. 2A). Peak
systolic pressure only differed between wild-type and transgenic hearts
during the initial few minutes of reperfusion, being almost identical
for the final 25 min of reperfusion (Fig. 2B). Thus, largely
as a result of the difference in diastolic dysfunction, left
ventricular developed pressure recovered gradually to only 55 ± 7 mmHg in wild-type hearts and almost completely to 94 ± 6 mmHg in
transgenic hearts (Fig. 2C). This latter value for
transgenic hearts did not differ significantly from preischemia. As
shown in Fig. 2C, the differences in contractile recovery between wild-type and transgenic hearts were evident not only
during the later stages of reperfusion (i.e.. from 5-30 min of
reperfusion) but also immediately on reperfusion and before recommencing electrical pacing.
|
|
|
dP/dt)
are shown in Fig. 4. All variables
responded similarly to ischemia-reperfusion, with final recoveries
being twofold higher in transgenic vs. wild-type hearts.
|
|
33.4 (±4.5) mmHg + (1.8 (±0.2) mmHg/min of reperfusion).
For transgenic hearts, recovery of left ventricular developed pressure
was calculated as developed pressure equal to 8.0 (±3.2 mmHg) + 1.9 (±0.2) mmHg/min of reperfusion.
The slopes (or rates) for recovery were not statistically different
(P > 0.05), whereas the intercepts differed
significantly (P < 0.05). Thus recoveries for
developed pressure are calculated to be parallel in wild-type and
transgenic hearts, differing by a constant value of ~25 mmHg
throughout the final 20-25 min of reperfusion.
Effects of KATP blockade with 5-HD. Preischemic function (end-diastolic pressure, left ventricular developed pressure, coronary flow) was not significantly altered by 5-HD pretreatment (Figs. 2 and 3). Blockade of KATP channels with 5-HD had no significant effect on time to onset of contracture or degree of contracture during the initial ischemic insult in wild-type hearts, but significantly increased the rapidity of ischemic contracture in transgenic hearts (Fig. 1). 5-HD reduced contractile recovery during reperfusion in both groups (Figs. 2 and 4). The marked improvement in early recovery of diastolic and systolic pressures with A1AR overexpression (i.e., at 1-2 min of reperfusion) was essentially abolished by 5-HD treatment. 5-HD significantly elevated diastolic pressure during the first 5 min of reperfusion in wild-type hearts, but significantly and markedly elevated diastolic pressure throughout the entire reperfusion period in transgenic hearts (Fig. 2A). This effectively normalized the recovery of diastolic pressure between wild-type and transgenic hearts. 5-HD did not substantially alter early recovery of systolic pressure in wild-type hearts and only significantly depressed recovery at the final 30-min time point (Fig. 2B). Final recovery of systolic pressure was identical in wild-type and transgenic hearts treated with 5-HD.
Largely as a result of worsened diastolic dysfunction, 5-HD reduced the recovery of left ventricular developed pressure throughout the final 25 min of reperfusion in wild-type hearts (Fig. 2C). In transgenic hearts, 5-HD reduced recovery of developed pressure at every time point throughout reperfusion. Final recovery of developed pressure was 13 ± 6 mmHg in wild-type hearts and 24 ± 4 mmHg in transgenic hearts. Again, differences in contractile recovery were evident even during the initial 1-2 min of reperfusion and before commencement of electrical pacing (see Fig. 2C). 5-HD treatment significantly reduced coronary flow during the initial 5 min of reperfusion in both wild-type and transgenic hearts (Fig. 3). To assess the potential importance of this reduced hyperemia, we perfused a group of 5-HD-treated wild-type hearts at the same coronary flows observed in the untreated group (see Fig. 3). Functional recovery in these hearts did not differ at any time from values in 5-HD-treated hearts permitted intrinsic coronary flow (data not shown). Final recoveries for rate-pressure product, +dP/dt anddP/dt, were almost identical in both groups of
5-HD-treated wild-type hearts (Fig. 4). Although the marked
cardioprotection afforded by A1AR overexpression was
largely eliminated by 5-HD treatment (Fig. 2), recoveries for left
ventricular developed pressure, +dP/dt and dP/dt, remained slightly
but significantly higher in 5-HD-treated transgenic vs. 5-HD-treated
wild-type hearts (Figs. 2 and 4).
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The purpose of this study was to test the hypotheses that 1) intrinsic activation of mitochondrial KATP channels enhances tolerance to ischemia-reperfusion in murine heart and 2) activation of mitochondrial KATP channels contributes to protection afforded by A1AR overexpression. The cardioprotective roles of both KATP channels and A1ARs have received considerable attention in recent years. KATP activation is functionally and metabolically beneficial during ischemia-reperfusion (2, 7, 11, 13, 14, 16-18, 29, 30) and may be involved in preconditioning (1, 3, 4, 18, 21, 25, 30, 37, 41). There is also evidence that KATP channels are involved in cardioprotective effects of exogenous A1- and A3-adenosine receptor agonists (18, 25, 39-41). Examining the impact of a selective mitochondrial KATP blocker in wild-type hearts (i.e., blocking endogenously activated channels) and in hearts with 250-fold overexpression of A1ARs (i.e., blocking channels in the presence of amplified A1AR stimulation), we obtain evidence that KATP channel activation by intrinsic signals protects murine myocardium from ischemia-reperfusion. However, data reveal unique effects of KATP channels in the two different models studied.
The cardioprotective role of KATP channels in wild-type hearts. Although blockade of KATP channels reduces protective effects of preconditioning and exogenous A1AR activation in rat, guinea pig, rabbit, dog, and pig (1, 3, 4, 8, 18, 21, 25, 30, 37, 39-41), effects of KATP channels in the absence of such interventions are less clear. Some studies suggest that intrinsically activated KATP channels protect from ischemia and/or reperfusion (7, 8, 11, 30, 31), whereas others document no effects of KATP blockade under similar conditions (1, 4, 13, 16, 17, 36, 38). The impact of KATP activation or inhibition in the murine model has not been assessed. To unmask the role of intrinsically activated KATP channels, we used the mitochondrial KATP blocker 5-HD (3, 17, 21, 25, 26, 29, 37, 40, 41). Recent studies implicate mitochondrial rather than sarcolemmal KATP channels in cardioprotection (4, 12, 14, 22, 26), and 5-HD is known to block protection afforded by both selective and nonselective mitochondrial KATP openers (12, 15, 26, 29, 36) without modifying sarcolemmal KATP responses in cardiac or vascular tissue (29). 5-HD also inhibits anti-infarct effects of preconditioning (1, 3, 21, 25, 37, 41) and A1AR activation (25, 40, 41).
5-HD did not alter baseline function or flow in mouse heart, in agreement with studies in other species (3, 21, 29, 41). However, 5-HD markedly depressed contractile recovery from ischemia (Figs. 2 and 4). These are the first data indicating that KATP channels enhance ischemic tolerance in murine myocardium. Effects of KATP blockade were largely restricted to diastolic function, only modifying systolic pressure at the final 30 min time point (Fig. 2). Cardioprotection via intrinsically activated KATP channels therefore appears directed at improvement of diastolic function. Interestingly, although postischemic recovery was impaired by KATP blockade, the severity of ischemic contracture was unaltered (Fig. 2). It might be argued that 5-HD does not effectively block channels during ischemia. However, contracture was accelerated by 5-HD in transgenic hearts (Fig. 1), verifying the drug's ability to act during ischemia. A lack of effect of KATP blockade on contracture agrees with some studies in other species (1, 16, 17, 20, 29, 36) but contrasts with others (7, 11, 30, 31). Unaltered ischemic contracture does not imply that KATP channels targeted by 5-HD are not activated during ischemia. As with delayed effects of preconditioning, preischemic or ischemic KATP activation may improve recovery at a later stage. However, the present data and previous observations argue against this possibility. In all species studied to date, exogenous KATP channel activation delays ischemic contracture (1, 4, 11-13, 16, 17, 30, 31). Therefore, unless mouse is the only species in which KATP activation fails to alter contracture, the lack of effect of 5-HD indicates that KATP channels are not sufficiently activated during ischemia to modify contracture. Additionally, the fact that KATP openers universally delay ischemic contracture (2, 11-13, 16, 17, 30, 31) demonstrates that KATP channels are normally minimally activated at this time, otherwise they would be refractory to applied activators. If KATP channels are not intrinsically activated during ischemia they will be unresponsive to antagonists, as shown here (Figs. 1 and 2), and responsive to exogenous KATP openers (2, 11-13, 16, 17, 30, 31). Similarly, our observation of sensitivity to 5-HD during reperfusion (Fig. 2) is consistent with a lack of effect of KATP openers during reperfusion (15) and argues for intrinsic activation of KATP channels during reperfusion rather than ischemia. Other studies show a lack of effect of KATP blockade on ischemic contracture (1, 13, 16, 17, 29, 36). We conclude that KATP channels are not endogenously activated sufficiently to modify contracture during ischemia and that the difference in contractile recovery is likely mediated by KATP activation during reperfusion. Reduced postischemic recovery with 5-HD in murine heart contrasts with some studies in rat in which glibenclamide or 5-HD fails to alter postischemic function (13, 16, 17, 29, 36). The major drawback to these earlier studies is extremely poor postischemic recovery in control hearts (as little as 4-7% recovery of developed pressure) (16, 17), falling well below predicted levels for rat hearts subjected to 20-30 min normothermic ischemia (40-70% recovery) (9, 10, 27). The extremely low recovery in these studies precludes detection of any detrimental effects of KATP blockers. In studies in which postischemic recovery is more appropriate, variable responses to KATP blockers are observed. Some find that low levels of glibenclamide (0.3-10 µM) do not alter postischemic function (7, 11, 38), whereas others find that higher doses (30-50 µM) do depress recovery (8, 31). Reasons for discrepancies in effects of KATP blockade are unclear but may involve abnormally low recoveries (e.g., Refs. 13, 16, 17, 29, 36) and the possibility that low concentrations of glibenclamide (e.g., Refs. 7, 11, 38) are less effective in inhibiting the mitochondrial channels involved in KATP-mediated protection (4, 12, 14, 22, 26). Our data, together with previous studies, shed light on mechanisms by which KATP activation enhances recovery. Earlier studies uniformly demonstrate that 5-HD fails to modify necrosis in the absence of A1AR agonists or preconditioning (3, 4, 25, 26, 37, 40). It is therefore unlikely that changes in diastolic function observed with 5-HD (Fig. 2A) reflect tissue necrosis. This is borne out by normal recovery of peak systolic function throughout the first 25 min of reperfusion in 5-HD-treated hearts (Fig. 2B). Selective effects of 5-HD on diastolic vs. systolic function (Fig. 2) and the lack of effect of 5-HD on tissue necrosis in other species (3, 4, 25, 26, 37, 40) collectively argue for KATP-dependent attenuation of postischemic diastolic dysfunction. Another possible mechanism for reduced recovery is impaired coronary vasodilation (6, 35). We observed a modest 5-HD-dependent reduction in flow on reperfusion (Fig. 3). Because 5-HD does not modify vascular KATP channels and responses (15, 29), it is likely that this is secondary to enhanced postischemic contracture and vascular compression. Indeed, we show that manually perfusing 5-HD-treated hearts at higher flows observed in control hearts failed to reduce the impact of 5-HD on contractile recovery (Fig. 4).Cardioprotection with A1AR overexpression. We presented preliminary data demonstrating cardioprotection with A1AR overexpression in empty hearts with function assessed by apical displacement (28, 32). Additionally, we recently showed in the apicobasal model that A1AR overexpression reduces tissue necrosis, as indicated by inhibition of postischemic enzyme efflux and infarct size (32). Although the apicobasal model has been studied by other groups (33, 34, 42), metabolic rate is reduced and cellular energy state is elevated in empty vs. isovolumically functioning hearts (20). Because responses to ischemia-reperfusion are dependent on preischemic metabolic rate, it is relevant to examine the impact of A1AR overexpression in isovolumically contracting hearts developing higher wall stresses. The isovolumic Langendorff perfused heart is the most common ex vivo model employed in studies of ischemia-reperfusion in larger species (9, 10, 27). Our data show that A1AR overexpression markedly improves tolerance to ischemia-reperfusion in the isovolumic model (Figs. 1, 2, and 4). Transgenic hearts showed improved responses to both ischemia and reperfusion. On the basis of the pattern of changes observed, we suggest that A1AR overexpression primarily attenuates ischemic rather than reperfusion injury, consistent with A1AR inhibition of ischemic injury in other species (23-25). This is supported by a reduction in rapidity and severity of ischemic contracture, marked enhancement of contractile function immediately on reperfusion, and equivalent rates of contractile recovery during the final stages of reperfusion in wild-type and transgenic hearts.
Immediate contractile recovery during reperfusion is indicative of the severity of injury incurred during the prior ischemic insult, with subsequent changes in function reflecting onset and added consequences of reperfusion injury (27). A1AR overexpression substantially improved immediate recovery on reperfusion (Fig. 2), indicating reduced ischemic injury. Analysis of postischemic function reveals that after the initial 5-10 min of reperfusion, the rate of recovery is identical in wild-type and transgenic hearts (2 mmHg/min), the only difference being initial contractile recovery that differs by ~25 mmHg and is a reflection of ischemic damage (27). Reduced contracture development and the pattern of recovery during reperfusion support a reduction in ischemic rather than reperfusion injury in transgenic hearts. Extent of cardioprotection (i.e., 100% improvement in contractile recovery after 20 min global normothermic ischemia and abolition of postischemic contracture) is equal to or greater than protection after overexpression of glutathione peroxidase (42), superoxide dismutase (5), and heat shock proteins (33, 34).Role of KATP channels in transgenic hearts overexpressing A1ARs. The precise mechanisms contributing to protection with A1AR overexpression remain unclear. In initial studies, we hypothesized that KATP channels may contribute (19). A1ARs activate KATP channels (22, 25), exogenous activation of KATP channels or A1ARs produce similar functional and metabolic effects during ischemia-reperfusion (7, 11-13, 17-19, 23-25, 30), and KATP blockade attenuates cardioprotection afforded by A1AR agonists (25, 39-41). We show here that 5-HD eliminates cardioprotection in hearts overexpressing A1ARs: recoveries are comparable in 5-HD-treated wild-type and transgenic hearts (Figs. 1, 2, and 4). Specific effects of A1AR overexpression were all inhibited by 5-HD: A1AR overexpression reduced ischemic contracture and 5-HD eliminated this effect without altering contracture in wild-type hearts (Fig. 1); A1AR overexpression improved systolic recovery during the first minutes of reperfusion (Fig. 2B), and 5-HD eliminated this effect without altering recovery in wild-type hearts; and A1AR overexpression improved recovery of diastolic pressure throughout reperfusion (Fig. 2A), and 5-HD also eliminated this effect. Our data are consistent with a role for KATP channels in cardioprotection with A1AR overexpression and with 5-HD-dependent reductions in protective effects of exogenous adenosine agonists (25, 39, 41).
Although the KATP blocker worsened contracture in transgenic hearts, it failed to alter contracture in wild-type hearts (Fig. 1). This observation is entirely consistent with the lack of effect of 5-HD or glibenclamide on ischemic contracture in the absence of exogenous A1AR agonists (2, 7, 11, 13, 16, 17, 29, 36, 38) and selective inhibition of the effects of exogenous A1AR activation (25, 38, 40). The lack of effect of 5-HD in wild-type hearts and acceleration of contracture in transgenic hearts supports the conclusion that KATP channels only modulate ischemic contracture when they are maximally or near maximally activated, for example by overexpression of A1ARs (Fig. 1) or application of A1AR agonists (24, 39, 40). In conclusion, this study is the first to examine the role of KATP channels in murine myocardium. Our findings demonstrate that 1) intrinsic activation of mitochondrial KATP channels enhances recovery from ischemia without modifying rapidity or severity of ischemic contracture, 2) effects of intrinsic KATP activation are directed at improving recovery of diastolic rather than systolic function during reperfusion, 3) ~250-fold A1AR overexpression delays and reduces injury during ischemia and improves contractile recovery on reperfusion, and 4) KATP channel activation plays a role in cardioprotection mediated by overexpressed A1ARs. We conclude that intrinsic activation of mitochondrial KATP channels is an important cardioprotective mechanism in murine myocardium and appears to be beneficially activated by endogenous signals during reperfusion rather than ischemia.| |
ACKNOWLEDGEMENTS |
|---|
The authors acknowledge the excellent technical assistance of Anne Byford.
| |
FOOTNOTES |
|---|
This work was supported in part by the University of Virginia Children's Medical Center, the Children's Heart Foundation, the American Heart Association Virginia Affiliate, and a grant from the National Health and Medical Research Council of Australia. Drs. Gauthier and Morrison were supported by American Heart Association Virginia Affiliate research fellowships and Dr. Matherne was supported by an American Heart Association Established Investigator Grant.
Address for reprint requests and other correspondence: J. P. Headrick, Rotary Centre for Cardiovascular Research, Griffith Univ., Gold Coast, Southport, QLD 4217 Australia (E-mail: j.headrick{at}mailbox.gu.edu.au).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 5 April 1999; accepted in final form 4 April 2000.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Armstrong, SC,
Kao R,
Gao W,
Shivell LC,
Downey JM,
Honkanen RE,
and
Ganote CE.
Comparison of in vitro preconditioning responses of isolated pig and rabbit cardiomyocytes: effects of a protein phosphatase inhibitor, fostriecin.
J Mol Cell Cardiol
29:
3009-3024,
1997[ISI][Medline].
2.
Armstrong, SC,
Liu GS,
Downey JM,
and
Ganote CE.
Potassium channels and preconditioning of isolated rabbit cardiomyocytes: effects of glyburide and pinacadil.
J Mol Cell Cardiol
27:
1765-1774,
1995[ISI][Medline].
3.
Auchampach, JA,
Grover GJ,
and
Gross GJ.
Blockade of ischaemic preconditioning in dogs by the novel ATP dependent potassium channel antagonist sodium 5-hydroxydecanoate.
Cardiovasc Res
26:
1054-1062,
1992
4.
Baines, CP,
Liu GS,
Birincioglu M,
Critz SD,
Cohen MV,
and
Downey JM.
Ischemic preconditioning depends on interaction between mitochondrial KATP channels and actin cytoskeleton.
Am J Physiol Heart Circ Physiol
276:
H1361-H1368,
1999
5.
Chen, EP,
Bittner HB,
Davis D,
Folz RJ,
and
Van Trigt P.
Extracellular superoxide dismutase transgene overexpression preserves postischemic myocardial function in isolated murine hearts.
Circulation
94:
II412-II417,
1996.
6.
Daut, J,
Maier-Rudolph W,
Von Beckerath N,
Mehrke G,
Gunther K,
and
Goedle-Meinen L.
Hypoxic dilation of coronary arteries is mediated by ATP-sensitive potassium channels.
Science
247:
1341-1344,
1990
7.
Docherty, JC,
Gunter HE,
Kuzio B,
Shoemaker L,
Yang L,
and
Deslauriers R.
Effects of cromakalim and glibenclamide on myocardial high energy phosphates and intracellular pH during ischemia-reperfusion: 31P NMR studies.
J Mol Cell Cardiol
29:
1665-1673,
1997[ISI][Medline].
8.
Ford, WR,
Lopaschuk GD,
Schulz R,
and
Clanachan AS.
KATP-channel activation: effects on myocardial recovery from ischaemia and role in the cardioprotective response to adenosine A1-receptor stimulation.
Br J Pharmacol
124:
639-646,
1998[ISI][Medline].
9.
Galinanes, M,
and
Hearse DJ.
Assessment of ischemic injury and protective interventions: the Langendorff versus the working rat heart preparation.
Can J Cardiol
6:
83-91,
1990[ISI][Medline].
10.
Galinanes, M,
and
Hearse DJ.
Species differences in susceptibility to ischemic injury and responsiveness to myocardial protection.
Cardioscience
1:
127-143,
1990[ISI][Medline].
11.
Galinanes, M,
Shattock MJ,
and
Hearse DJ.
Effects of potassium channel modulation during global ischaemia in isolated rat heart with and without cardioplegia.
Cardiovasc Res
26:
1063-1068,
1992[ISI][Medline].
12.
Garlid, KD,
Paucek P,
Yarov-Yarovoy V,
Murray HN,
Darbenzio RB,
D'Alonzo AJ,
Lodge NJ,
Smith MA,
and
Grover GJ.
Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels. Possible mechanism of cardioprotection.
Circ Res
81:
1072-1082,
1997
13.
Grover, GJ.
Protective effects of ATP-sensitive potassium-channel openers in experimental myocardial ischemia.
J Cardiovasc Pharmacol
24:
S18-S27,
1994.
14.
Grover, GJ,
D'Alonzo AJ,
Hess T,
Sleph PG,
and
Darbenzio RB.
Glyburide-reversible cardioprotective effect of BMS-180448 is independent of action potential shortening.
Cardiovasc Res
30:
731-738,
1995[ISI][Medline].
15.
Grover, GJ,
Dzwonczyk S,
and
Sleph PG.
Reduction of ischemic damage in isolated rat hearts by the potassium channel opener RP 52891.
Eur J Pharmacol
191:
11-19,
1990[ISI][Medline].
16.
Grover, GJ,
McCullough JR,
Henry DE,
Conder ML,
and
Sleph PG.
Anti-ischemic effects of the potassium channel activators pinacidil and cromakalim and the reversal of these effects with the potassium channel blocker glyburide.
J Pharmacol Exp Ther
251:
98-104,
1989
17.
Grover, GJ,
Sleph P,
and
Dzwonczyk S.
Pharmacologic profile of cromakalim in the treatment of myocardial ischemia in isolated rat hearts and anesthetized dogs.
J Cardiovasc Pharmacol
16:
853-864,
1990[ISI][Medline].
18.
Grover, GJ,
Sleph P,
and
Dzwonczyk S.
Role of myocardial ATP-sensitive potassium channels in mediating preconditioning in the dog heart and their possible interaction with adenosine A1 receptors.
Circulation
86:
1310-1316,
1992
19.
Headrick, JP,
Gauthier NS,
Berr SS,
and
Matherne GP.
Transgenic A1 adenosine receptor overexpression improves myocardial energy state during ischemia reperfusion.
J Mol Cell Cardiol
30:
1059-1064,
1998[ISI][Medline].
20.
Headrick, JP,
Matherne GP,
Berr SS,
and
Berne RM.
Effects of graded perfusion and isovolumic work on epicardial and venous adenosine and cytosolic metabolism.
J Mol Cell Cardiol
23:
309-324,
1991[ISI][Medline].
21.
Hide, EJ,
and
Thiemermann C.
Limitation of myocardial infarct size in the rabbit by ischaemic preconditioning is abolished by sodium 5-hydroxydecanoate.
Cardiovasc Res
31:
941-946,
1996[ISI][Medline].
22.
Holmuhamedov, EL,
Jovanovic S,
Dzeja PP,
Jovanovic A,
and
Terzic A.
Mitochondrial ATP-sensitive K+ channels modulate cardiac mitochondrial function.
Am J Physiol Heart Circ Physiol
275:
H1567-H1576,
1998
23.
Lasley, RD,
and
Mentzer RM, Jr.
Adenosine improves recovery of postischemic myocardial function via an adenosine A1 receptor mechanism.
Am J Physiol Heart Circ Physiol
263:
H1460-H1465,
1992
24.
Lasley, RD,
Rhee JW,
Van Wylen DGL,
and
Mentzer RM, Jr.
Adenosine A1 receptor mediated protection of the globally ischemic isolated rat heart.
J Mol Cell Cardiol
22:
29-47,
1990.
25.
Liang, BT.
Direct preconditioning of cardiac ventricular myocytes via adenosine A1 receptors and KATP channel.
Am J Physiol Heart Circ Physiol
271:
H1769-H1777,
1996
26.
Liu, Y,
Sato T,
O'Rourke B,
and
Marban E.
Mitochondrial ATP-dependent potassium channels. Novel effectors of cardioprotection?
Circulation
97:
2463-2469,
1998
27.
Manche, A,
Edmondson SJ,
and
Hearse DJ.
Dynamics of early post-ischemic myocardial functional recovery.
Circulation
92:
526-534,
1995
28.
Matherne, GP,
Linden J,
Byford AM,
Gauthier NS,
and
Headrick JP.
Transgenic A1 adenosine receptor over-expression increases myocardial resistance to ischemia.
Proc Natl Acad Sci USA
94:
6541-6546,
1997
29.
McCullough, JR,
Normandin DE,
Conder ML,
Sleph PG,
Dzwonczyk S,
and
Grover GJ.
Specific block of the anti-ischemic actions of cromakalim by sodium 5-hydroxydecanoate.
Circ Res
69:
949-958,
1991
30.
McPherson, CD,
Pierce GN,
and
Cole WC.
Ischemic cardioprotection by ATP-sensitive K+ channels involves high-energy phosphate preservation.
Am J Physiol Heart Circ Physiol
265:
H1809-H1818,
1993
31.
Mitani, A,
Kinoshita K,
Fukamachi K,
Sakamoto M,
Kurisu K,
Tsuruhara Y,
Fukumura F,
Nakashima A,
and
Tokunaga K.
Effects of glibenclamide and nicorandil on cardiac function during ischemia and reperfusion in isolated perfused rat hearts.
Am J Physiol Heart Circ Physiol
261:
H1864-H1871,
1991
32.
Morrison, RR,
Jones R,
Byford AM,
Stell AR,
Peart J,
Headrick JP,
and
Matherne GP.
Transgenic overexpression of cardiac A1 adenosine receptors mimics ischemic preconditioning.
Am J Physiol Heart Circ Physiol
279:
H1071-1078,
2000
33.
Plumier, JCL,
Ross BM,
Currie RW,
Angelidis CE,
Kazlaris H,
Kollias G,
and
Pagoulatos GN.
Transgenic mice expressing the human heat shock protein 70 have improved post-ischemic myocardial recovery.
J Clin Invest
95:
1854-1860,
1995.
34.
Radford, NB,
Fina M,
Benjamin IJ,
Moreadith RW,
Graves KH,
Zhao P,
Gavva S,
Wiethoff A,
Sherry AD,
and
Malloy CR.
Cardioprotective effects of 70-kDa heat shock protein in transgenic mice.
Proc Natl Acad Sci USA
93:
2339-2342,
1996
35.
Samaha, FF,
Heineman FW,
Ince C,
Fleming J,
and
Balaban RS.
ATP-sensitive potassium channel is essential to maintain basal coronary vascular tone in vivo.
Am J Physiol Cell Physiol
262:
C1220-C1227,
1992
36.
Sargent, CA,
Smith MA,
Dzwonczyk S,
Sleph PG,
and
Grover GJ.
Effect of potassium channel blockade on the anti-ischemic actions of mechanistically diverse agents.
J Pharmacol Exp Ther
259:
97-103,
1991
37.
Schulz, JE,
Qian YZ,
Gross GJ,
and
Kukreja RC.
The ischemia-selective KATP channel antagonist, 5-hydroxydecanoate, blocks ischemic preconditioning in the rat heart.
J Mol Cell Cardiol
29:
1055-1060,
1997[ISI][Medline].
38.
Thourani, VH,
Nakamura M,
Ronson RS,
Jordan JE,
Zhao ZQ,
Levy JH,
Szlam F,
Guyton RA,
and
Vinten-Johansen J.
Adenosine A3-receptor stimulation attenuates postischemic dysfunction through KATP channels.
Am J Physiol Heart Circ Physiol
277:
H228-H235,
1999
39.
Toombs, CF,
McGee DS,
Johnston WE,
and
Vinten-Johansen J.
Protection from ischaemic-reperfusion injury with adenosine pretreatment is reversed by inhibition of ATP sensitive potassium channels.
Cardiovasc Res
27:
623-629,
1993[ISI][Medline].
40.
Van Winkle, DM,
Chien LG,
Wolff RA,
Soifer BE,
Kuzume K,
and
Davis RF.
Cardioprotection afforded by adenosine receptor activation is abolished by blockade of the KATP channel.
Am J Physiol Heart Circ Physiol
266:
H829-H839,
1994
41.
Yao, Z,
and
Gross G.
A comparison of adenosine-induced cardioprotection and ischemic preconditioning in dogs. Efficacy, time course, and role of KATP channels.
Circulation
89:
1229-1236,
1994
42.
Yoshida, T,
Watanabe M,
Engelman DT,
Engelman RM,
Schley JA,
Maulik N,
Ho YS,
Oberley TD,
and
Das DK.
Transgenic mice overexpressing glutathione peroxidase are resistant to myocardial ischemia reperfusion injury.
J Mol Cell Cardiol
28:
1759-1767,
1996[ISI][Medline].
This article has been cited by other articles:
|
|
 |
|
  J. P. Headrick, B. Hack, and K. J. Ashton Acute adenosinergic cardioprotection in ischemic-reperfused hearts Am J Physiol Heart Circ Physiol, November 1, 2003; 285(5): H1797 - H1818. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Zaugg, E. Lucchinetti, M. Uecker, T. Pasch, and M. C. Schaub Anaesthetics and cardiac preconditioning. Part I. Signalling and cytoprotective mechanisms Br. J. Anaesth., October 1, 2003; 91(4): 551 - 565. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. N. Peart and G. J. Gross Adenosine and opioid receptor-mediated cardioprotection in the rat: evidence for cross-talk between receptors Am J Physiol Heart Circ Physiol, June 5, 2003; 285(1): H81 - H89. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Peart and J. P Headrick Adenosine-mediated early preconditioning in mouse: protective signaling and concentration dependent effects Cardiovasc Res, June 1, 2003; 58(3): 589 - 601. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. E. Regan, M. Broad, A. M. Byford, A. R. Lankford, R. J. Cerniway, M. W. Mayo, and G. P. Matherne A1 adenosine receptor overexpression attenuates ischemia-reperfusion-induced apoptosis and caspase 3 activity Am J Physiol Heart Circ Physiol, March 1, 2003; 284(3): H859 - H866. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. J Ashton, K. Holmgren, J. Peart, A. R Lankford, G Paul Matherne, S. Grimmond, and J. P Headrick Effects of A1 adenosine receptor overexpression on normoxic and post-ischemic gene expression Cardiovasc Res, March 1, 2003; 57(3): 715 - 726. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Peart, L. Willems, and J. P. Headrick Receptor and non-receptor-dependent mechanisms of cardioprotection with adenosine Am J Physiol Heart Circ Physiol, February 1, 2003; 284(2): H519 - H527. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Han, N. Kim, H. Joo, and E. Kim Ketamine abolishes ischemic preconditioning through inhibition of KATP channels in rabbit hearts Am J Physiol Heart Circ Physiol, July 1, 2002; 283(1): H13 - H21. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. H. Talukder, R. R. Morrison, M. A. Jacobson, K. A. Jacobson, C. Ledent, and S. J. Mustafa Targeted deletion of adenosine A3 receptors augments adenosine-induced coronary flow in isolated mouse heart Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2183 - H2189. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. H. Talukder, R. R. Morrison, and S. J. Mustafa Comparison of the vascular effects of adenosine in isolated mouse heart and aorta Am J Physiol Heart Circ Physiol, January 1, 2002; 282(1): H49 - H57. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Peart, G. Paul Matherne, R. J. Cerniway, and J. P. Headrick Cardioprotection with adenosine metabolism inhibitors in ischemic-reperfused mouse heart Cardiovasc Res, October 1, 2001; 52(1): 120 - 129. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Yang, R. J. Cerniway, A. M. Byford, S. S. Berr, B. A. French, and G. P. Matherne Cardiac overexpression of A1-adenosine receptor protects intact mice against myocardial infarction Am J Physiol Heart Circ Physiol, March 1, 2002; 282(3): H949 - H955. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. H. Talukder, R. R. Morrison, M. A. Jacobson, K. A. Jacobson, C. Ledent, and S. J. Mustafa Targeted deletion of adenosine A3 receptors augments adenosine-induced coronary flow in isolated mouse heart Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2183 - H2189. [Abstract] [Full Text] [PDF]
|
|
| ||||
P < 0.05, 5-HD-treated vs.
untreated hearts.
, 
) and transgenic (
,
) mouse hearts. Hearts were either untreated
(
