The effect of eplerenone on ischemia reperfusion injury in human myocardium

Coronary heart disease is the leading cause of death worldwide. Rapid
myocardial reperfusion is the most effective strategy to limit infarct size in
patients with a myocardial infarction. Paradoxically, the process of
reperfusion of ischemic myocardium itself can also aggravate injury
(*reperfusion injury*). Therefore, mortality and morbidity of patients with a
myocardial infarction remain high, and novel strategies to reduce
ischemia-reperfusion (IR) injury are urgently needed.

It has been suggested that the mineralocorticoid receptor (MR) antagonists
spironolactone and eplerenone could potentially serve this goal, since
treatment with these drugs reduces cardiovascular mortality in patients with
heart failure (1). This hypothesis was recently confirmed in murine models of
myocardial infarction, which demonstrated that the administration of MR
antagonists, either immediately before the onset of ischemia, or at the moment
of reperfusion, profoundly reduced infarct size (2,3). Interestingly, this
cardioprotective effect was also observed in hearts from adrenalectomized rats
(2), suggesting that this effect is not merely due to competitive antagonism of
endogenous aldosterone, but could also be because spironolactone can function
as an inverse agonist of the MR.

The underlying mechanism of the cardioprotective effect is currently unknown,
but a recent study suggests the involvement of adenosine receptor stimulation
(3). Adenosine is an endogenous purine nucleoside, which is known to increase
the tolerance of various tissues, including the myocardium and blood vessels,
against ischemia and reperfusion (4). The extracellular enzyme ecto-5*-
nucleotidase (also named CD73) is the rate-limiting enzyme in the extracellular
formation of adenosine from adenosine monophosphate (AMP). Interestingly, in a
recent study in mice, the cardioprotective effect of MR antagonists was
abolished after targeted gene deletion of CD73. This observation suggests that
adenosine is a key mediator of this effect. Interestingly, it has recently been
shown that statins limit infarct size also by activation of myocardial CD73.
The cardioprotective effects of MR antagonists have so far only been studied in
isolated cells and in a few murine models of myocardial infarction.

In the current research proposal, we aim to translate these preclinical
findings to the human situation and test the hypothesis that MR antagonists
limits IR-injury in human myocardial tissue. Secondly, we will test the
hypothesis that the cardioprotective effect is mediated by adenosine receptor
stimulation. Elucidation of this underlying signaling cascade is crucial to
guarantee optimal future use of these drugs in clinical practice: if the
cardioprotective effect is indeed mediated by increased extracellular formation
of adenosine, then concomitant use of the adenosine receptor antagonist
caffeine or theophylline will abolish this effect, whereas the adenosine uptake
blocked dipyridamole could potentiate this effect.

This study is an ex vivo study on human atrial tissue that is harvested during
coronary bypass surgery.

In patients undergoing coronary artery bypass surgery, prior to connection to
the extracorporal circulation, the right auricle is harvested by the
cardiothoracic surgeon.

Immediately after excision, the auricle will be placed in a cold (4° C)
modified Tyrode*s solution, that is continuously gassed with 95% O2 and 5% CO2,
as described previously (6). Subsequently, two atrial trabeculae will be
isolated, suspended in organ bath, and connected to a force transducer.
Contraction will be induced by electrical field stimulation. Ischemia and
reperfusion will be simulated by superfusing the trabeculae with substrate-free
and hypoxic modified Tyrode*s solution and rapid pacing at 3 Hz. The
superfusate will be pumped into an artificial lung filled with 95% N2/5% CO2 to
obtain a low pO2. The recovery of contractile force after 90 minutes of
simulated ischemia and 120 minutes of reperfusion with oxygenated buffer will
be used as endpoint of ischemia reperfusion injury. Using this experimental
design, we have previously shown that a brief period of ischemia immediately
before the prolonged period of simulated ischemia augments recovery of
contractile function during reperfusion, which is called *ischemic
preconditioning* (6).

Experiment 1:
In vitro two trabeculae (diameter <1 mm; length >3 mm) will be dissected from
the atrial appendage, vertically suspended in an organ bath, linked to a force
transducer, and superfused with pre-oxygenated Tyrode*s buffer (pO2 500 to 600
mm Hg). Electrical field stimulation will be applied to induce contraction.
After equilibration, a baseline recording is performed during 20 min. Those
trabeculae that fail to produce at least 0.2 g of developed force at the end of
baseline or in which the coefficient of variation of developed force exceeded
20% are excluded.
Immediately after baseline recordings, for each patient the 2 trabeculae are
randomly assigned to either a stimulus for IP or continued superfusion with
Tyrode*s solution, so that from each patient 1 trabecula is preconditioned and
the other was not. Ischemic preconditioning is induced by 5 min of simulated
ischemia and 5 min of simulated reperfusion, as previously described (6). We
will also start with this experiment in the current research proposal as a
positive control, to ensure that the model is still working properly and it is
possible to limit IR injury in this setting. Simulated ischemia is accomplished
by superfusing the trabeculae with substrate-free modified Tyrode*s solution
(7.0 mM choline chloride substituted for glucose and pyruvate) and rapid pacing
at 3 Hz. The superfusate is pumped into an artificial lung filled with 95%
N2/5% CO2, which results in a low pO2 of 10 to 20 mm Hg. Subsequently, both
trabeculae are subjected to 90 min of simulated ischemia and 120 min of
simulated reperfusion. The percentage recovery (compared to second baseline) of
contractile force of the trabeculae at the end of reperfusion (last 10 minutes)
will serve as the primary endpoint. Should we not be able to reproduce our
previous results that ischemic preconditioning augments recovery of contractile
function, than the experiment will be stopped.

Experiment 2:
In the next 10 patients, a similar ischemia-reperfusion experiment will be
performed, but now the two trabeculae from each patient will be randomized to
either pretreatment with eplerenone (dissolved in DMSO, final concentration <
0.01%) or DMSO (vehicle), which will be present in the organ bath throughout
the experiment. Eplerenone will be administered in a final concentration of 10
µmol/l in the organ bath, as this has been shown to limit myocardial infarct
size in a previous study (3). This concentration is a relevant concentration,
since pharmacokinetic studies in humans have shown that the plasma
concentration after oral administration of 100 mg of eplerenone is
approximately 3-6 µmol/l (6). Eplerenone will be added after 10 minutes of
baseline recording, which will continue for another 10 minutes to be able to
observe a direct effect of eplerenone on contractile force.

Experiment 3:
If the results of experiment 2 show a cardioprotective effect of eplerenone,
than experiment 3 will be performed to investigate whether this effect is
dependent on adenosine receptor stimulation. In this experiment the trabeculae
will be exposed to either eplerenone of eplerenone together with the adenosine
receptor antagonist caffeine. Caffeine will be administered in a final
concentration of 10 mg/l, which has previously been shown to significantly
block ischemic preconditioning in a similar experimental design (7).

Experiment 4: if eplerenone does not show a cardioprotective effect in
experiment 2, we will perform two additional studies:
4a: in this experiment, the trabeculae of 10 patients will be randomized to
either pretreatment with aldosterone or vehicle to study whether aldosterone
itself increases ischemia-reperfusion injury. Aldosterone will be administered
in a final concentration of 10 nmol/l (which is slightly higher than the plasma
aldosterone concentration in patients with primary hyperaldosteronism).
In addition experiment 4b will be performed in which experiment 2 will be
repeated in the presence of 0.5 ng/ml aldosterone, which is similar to the
normal circulating plasma concentration of aldosterone in humans.

The intervention occurs ex vivo in the organ bath: adminstration of eplerenone
with or without the adenosine receptor antagonist caffeine.

The removal of the right atrial appendage does not confer any risk to the
patients.