Myocardium tolerant to an adenosine-dependent ischemic preconditioning
stimulus can still be protected by stimuli that employ alternative
David A. Liem,1Maaike te Lintel Hekkert,1Olivier C. Manintveld,1
Frans Boomsma,2Pieter D. Verdouw,1and Dirk J. Duncker1
1Experimental Cardiology, Thoraxcenter, and2Internal Medicine, Cardiovascular Research
Institute COEUR, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
Submitted 31 August 2004; accepted in final form 11 October 2004
Liem, David A., Maaike te Lintel Hekkert, Olivier C. Manint-
veld, Frans Boomsma, Pieter D. Verdouw, and Dirk J. Duncker.
Myocardium tolerant to an adenosine-dependent ischemic precondi-
tioning stimulus can still be protected by stimuli that employ alterna-
tive signaling pathways. Am J Physiol Heart Circ Physiol 288:
H1165–H1172, 2005. First published October 14, 2004; doi:10.1152/
ajpheart.00899.2004.—Clinical studies on cardioprotection by prein-
farct angina are ambiguous, which may involve development of
tolerance to repeated episodes of ischemia. Not all preconditioning
stimuli use identical signaling pathways, and because patients likely
experience varying numbers of episodes of preinfarct angina of
different degrees and durations, it is important to know whether
myocardium tolerant to a particular preconditioning stimulus can still
be protected by stimuli employing alternative signaling pathways. We
tested the hypothesis that development of tolerance to a particular
stimulus does not affect cardioprotection by stimuli that employ
different signaling pathways. Anesthetized rats underwent classical,
remote or pharmacological preconditioning. Infarct size (IS), pro-
duced by a 60-min coronary artery occlusion (CAO), was determined
after 120 min of reperfusion. Preconditioning by two 15-min periods
of CAO (2CAO15, an adenosine-dependent stimulus) limited IS from
69 ? 2% to 37 ? 6%, but when 2CAO15 was preceded by 4CAO15,
protection by 2CAO15 was absent (IS ? 68 ? 1%). This development
of tolerance coincided with a loss of cardiac interstitial adenosine
release, whereas two 15-min infusions of adenosine (200 ?g/min iv)
still elicited cardioprotection (IS ? 40 ? 4%). Furthermore, cardio-
protection was produced when 4CAO15 was followed by the ade-
nosine-independent stimulus 3CAO3 (IS ? 50 ? 8%) or the remote
preconditioning stimulus of two 15-min periods of mesenteric artery
occlusion (IS ? 49 ? 6%). In conclusion, development of tolerance
to cardioprotection by an adenosine-dependent preconditioning stim-
ulus still allows protection by pharmacological or ischemic stimuli
intervention employing different signaling pathways.
infarct size; remote preconditioning
ISCHEMIC PRECONDITIONING (IPC) is the most powerful means of
endogenous cardioprotection against irreversible cell injury in
the experimental animal (26, 31). However, clinical studies on
infarct size (IS) limitation by brief anginal episodes preceding
acute myocardial infarction are ambiguous (3, 4, 16, 27, 28,
44); such ambiguity has been attributed to a loss of cardiopro-
tection by ischemic preconditioning in the aging (1, 2, 3, 19) or
pathological (9, 12, 15, 18) heart. Another confounding factor
could be development of tolerance to IPC, i.e., the loss of
cardioprotection when the same preconditioning stimulus is
repetitively applied (6, 14, 32). For example, Cohen et al. (6)
demonstrated that in rabbits the cardioprotection produced by
a single 5-min coronary artery occlusion (CAO) followed by
10 min of reperfusion (1CAO5) was lost when the 5-min CAO
stimulus was applied at 30-min intervals for 8 h during 3 days.
In recent years, it has become apparent that not all precon-
ditioning stimuli employ the same signaling pathway to exert
their cardioprotective action (7, 10, 23, 24, 34). For instance, in
the rat, cardioprotection by a single 15-min CAO followed by
10 min of reperfusion (1CAO15) is adenosine dependent but
does not involve reactive oxygen species (ROS), whereas
cardioprotection by three cycles of 3 min of CAO interspersed
by 5 min of reperfusion (3CAO3) depends on ROS (24) but
does not involve adenosine (22, 23). The major aim of the
present study was therefore to investigate whether tolerance
that develops when the same IPC stimulus is applied repeti-
tively also implies tolerance to a stimulus that employs a
different signal transduction pathway. Hence, in the first part of
the study, we investigated whether tolerance to a particular
(adenosine-dependent) preconditioning stimulus also affects
cardioprotection by a stimulus that employs an alternative
(adenosine-independent) pathway. Myocardium can be precon-
ditioned by local myocardial ischemia, as well as by brief
ischemia in noncardiac tissue such as the small intestine,
kidneys, and skeletal muscle (5, 11, 25, 30), which, at least for
the small intestine, involves a neurogenic pathway (11, 25).
Hence, in the second part of the study, we investigated whether
cardioprotection by remote preconditioning via a 15-min mes-
enteric artery occlusion (MAO15) is affected by the develop-
ment of tolerance to a classical IPC stimulus.
Because tolerance to IPC has not been investigated in the rat,
we first established a model for the development of tolerance
on the basis of our experience with the adenosine-dependent
stimulus 1CAO15 in this species. Capitalizing on the observa-
tions by Vogt et al. (41), who showed in pigs that progressive
loss of adenosine production rendered myocardium tolerant to
protection by 10-min CAO but still responsive to exogenous
adenosine, we also investigated whether loss of adenosine
release also contributes to development of tolerance in the rat
heart and whether exogenous adenosine still induces protection
once tolerance has developed.
Address for reprint requests and other correspondence: D. J. Duncker,
Experimental Cardiology, Thoraxcenter, Erasmus MC, Univ. Medical Center
Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands (E-mail:
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.
Am J Physiol Heart Circ Physiol 288: H1165–H1172, 2005.
First published October 14, 2004; doi:10.1152/ajpheart.00899.2004.
0363-6135/05 $8.00 Copyright © 2005 the American Physiological Society http://www.ajpheart.orgH1165
cally on the duration of the stimulus and involves both A1 and A3
receptors. Cardiovasc Res 51: 701–708, 2001.
24. Liem DA, Verdouw PD, Mies R, te Lintel Hekkert M, and Duncker
DJ. Mechanism of ischemic preconditioning depends critically on the
stimulus (Abstract). Circulation 106: II-133, 2002.
25. Liem DA, Verdouw PD, Ploeg H, Kazim S, and Duncker DJ. Sites of
action of adenosine in interorgan preconditioning of the heart. Am J
Physiol Heart Circ Physiol 283: H29–H37, 2002.
26. Murry CE, Jennings RB, and Reimer KA. Preconditioning with ische-
mia: a delay of lethal cell injury in ischemic myocardium. Circulation 74:
27. Nakagawa Y, Ito H, Kitakaze M, Kusuoka H, Hori M, Kuzuya T,
Higashino Y, Fujii K, and Minamino T. Effect of angina pectoris on
myocardial protection in patients with reperfused anterior wall myocardial
infarction: retrospective clinical evidence of “preconditioning.” J Am Coll
Cardiol 25: 1076–1083, 1995.
28. Noda T, Minatoguchi S, Fujii K, Hori M, Ito T, Kanmatsuse K,
Matsuzaki M, Miura T, Nonogi H, Tada M, Tanaka M, and Fujiwara
H. Evidence for the delayed effect in human ischemic preconditioning:
Prospective Multicenter Study for Preconditioning in Acute Myocardial
Infarction. J Am Coll Cardiol 34: 1966–1974, 1999.
29. Patel HH, Moore J, Hsu AK, and Gross GJ. Cardioprotection at a
distance: mesenteric artery occlusion protects the myocardium via an
opioid-sensitive mechanism. J Mol Cell Cardiol 34: 1317–1323, 2002.
30. Pell TJ, Baxter GF, Yellon DM, and Drew GM. Renal ischemia
preconditions myocardium: role of adenosine receptors and ATP-sensitive
potassium channels. Am J Physiol Heart Circ Physiol 275: H1542–H1547,
31. Przyklenk K and Kloner RA. Ischemic preconditioning: exploring the
paradox. Prog Cardiovasc Dis 40: 517–547, 1998.
32. Sack S, Mohri M, Arras M, Schwarz ER, and Schaper W. Ischaemic
preconditioning—time course of renewal in the pig. Cardiovasc Res 27:
33. Schoemaker RG and van Heijningen CL. Bradykinin mediates cardiac
preconditioning at a distance. Am J Physiol Heart Circ Physiol 278:
34. Schulz R, Post H, Vahlhaus C, and Heusch G. Ischemic preconditioning
in pigs: a graded phenomenon: its relation to adenosine and bradykinin.
Circulation 98: 1022–1029, 1998.
35. Schwarz ER, Somoano Y, Hale SL, and Kloner RA. What is the
required reperfusion period for assessment of myocardial infarct size using
triphenyltetrazolium chloride staining in the rat? J Thromb Thrombolysis
10: 181–187, 2000.
36. Smolenski RT, Lachno DR, Ledingham SJ, and Yacoub MH. Deter-
mination of sixteen nucleotides, nucleosides and bases using high-perfor-
mance liquid chromatography and its application to the study of purine
metabolism in hearts for transplantation. J Chromatogr 527: 414–420,
37. Tang ZL, Dai W, Li YJ, and Deng HW. Involvement of capsaicin-
sensitive sensory nerves in early and delayed cardioprotection induced by
a brief ischaemia of the small intestine. Naunyn Schmiedebergs Arch
Pharmacol 359: 243–247, 1999.
38. Tomai F, Crea F, Chiariello L, and Gioffre PA. Ischemic precondition-
ing in humans: models, mediators, and clinical relevance. Circulation 100:
39. Tsuchida A, Thompson R, Olsson RA, and Downey JM. The anti-
infarct effect of an adenosine A1-selective agonist is diminished after
prolonged infusion as is the cardioprotective effect of ischaemic precon-
ditioning in rabbit heart. J Mol Cell Cardiol 26: 303–311, 1994.
40. Van den Doel MA, Gho BC, Duval SY, Schoemaker RG, Duncker DJ,
and Verdouw PD. Hypothermia extends the cardioprotection by is-
chaemic preconditioning to coronary artery occlusions of longer duration.
Cardiovasc Res 37: 76–81, 1998.
41. Vogt AM, Ando H, Arras M, and Elsasser A. Lack of adenosine causes
myocardial refractoriness. J Am Coll Cardiol 31: 1134–1141, 1998.
42. Weinbrenner C, Schulze F, Sarvary L, and Strasser RH. Remote
preconditioning by infrarenal aortic occlusion is operative via ?1-opioid
receptors and free radicals in vivo in the rat heart. Cardiovasc Res 61:
43. Yellon DM, Alkhulaifi AM, and Pugsley WB. Preconditioning the
human myocardium. Lancet 342: 276–277, 1993.
44. Zahn R, Schiele R, Schneider S, Gitt AK, Seidl K, Bossaller C, Schuler
G, Gottwik M, Altmann E, Rosahl W, and Senges J. Effect of prein-
farction angina pectoris on outcome in patients with acute myocardial
infarction treated with primary angioplasty (results from the Myocardial
Infarction Registry). Am J Cardiol 87: 1–6, 2001.
TOLERANCE TO ISCHEMIC PRECONDITIONING
AJP-Heart Circ Physiol • VOL 288 • MARCH 2005 • www.ajpheart.org