Ischaemic tolerance in aged mouse myocardium: the role of adenosine and effects of A1 adenosine receptor overexpression

Abstract

The genesis of the ischaemia intolerant phenotype in aged myocardium is poorly understood. We tested the hypothesis that impaired adenosine-mediated protection contributes to ischaemic intolerance, and examined whether this is countered by A1 adenosine receptor (A1AR) overexpression. Responses to 20 min ischaemia and 45 min reperfusion were assessed in perfused hearts from young (24 months) and moderately aged (1618 months) mice. Post-ischaemic contractility was impaired by ageing with elevated ventricular diastolic (32 ± 2 vs. 18 ± 2 mmHg in young) and reduced developed (37 ± 3 vs. 83 ± 6 mmHg in young) pressures. Lactate dehydrogenase (LDH) loss was exaggerated (27 ± 2 vs. 16 ± 2 IU g1in young) whereas the incidence of tachyarrhythmias was similar in young (15 ± 1 %) and aged hearts (16 ± 1 %). Functional analysis confirmed equipotent effects of 50 ¼m adenosine at A1 and A2 receptors in young and aged hearts. Nonetheless, while 50 ¼m adenosine improved diastolic (5 ± 1 mmHg) and developed pressures (134 ± 7 mmHg) and LDH loss (6 ± 2 IU g1) in young hearts, it did not alter these variables in the aged group. Adenosine did attenuate arrhythmogenesis for both ages (to 10 %). In contrast to adenosine, 50 ¼m diazoxide reduced ischaemic damage and arrhythmogenesis for both ages. Contractile and anti-necrotic effects of adenosine were limited by 100 ¼m 5-hydroxydecanoate (5-HD) and 3 ¼m chelerythrine. Anti-arrhythmic effects were limited by 5-HD but not chelerythrine. Non-selective (100 ¼m 8-sulfophenyltheophylline) and A1-selective (150 nm 8-cyclopentyl-1,3-dipropylxanthine) adenosine receptor antagonism impaired ischaemic tolerance in young but not aged hearts. Quantitative real-time PCR and radioligand analysis indicated that impaired protection is unrelated to changes in A1AR mRNA transcription, or receptor density (8 fmol mg1 protein in both age groups). However, A1AR overexpression improved tolerance for both ages, restoring adenosine-mediated protection. These data reveal impaired protection via exogenous and endogenous adenosine contributes to ischaemic intolerance with ageing. This is independent of A1AR expression, and involves ineffective activation of a 5-HD-/diazoxide-sensitive process. The effects of A1AR overexpression indicate that the age-related failure in signalling can be overcome.There is increasing evidence of a decline in myocardial tolerance to injury with ageing. A reduction in the 'intrinsic' tolerance to ischaemic insult is supported by data from animal models (Pahor et al. 1985; Frolkis et al. 1991; Misare et al. 1992; Lesnefsky et al. 1994; Tani et al. 1997; Headrick, 1998; Abete et al. 1999; Rosenfeldt et al. 2002) and humans (Mariani et al. 2000; Rosenfeldt et al. 2002). The molecular basis of this intolerant phenotype is unclear, but it may involve multiple alterations including mitochondrial abnormalities (Lesnefsky et al. 2001), impaired anti-oxidant responses (Boucher et al. 1998; Coombes et al. 2000; Lesnefsky et al. 2001), loss of proteasome function (Bulteau et al. 2002) and modified Ca2+ handling (Cain et al. 1998). One possibility receiving increased attention is impairment of intrinsic cardioprotective responses (Abete et al. 1996; Gao et al. 2000; Schulman et al. 2001; Lee et al. 2002). This may be a particularly important factor since it may impact directly on the therapeutic approach to ischaemic injury in aged hearts. It is increasingly evident that conventional therapeutic strategies developed through findings in young tissues and subjects may not be relevant in aged subjects (Rosenfeldt et al. 2002).Adenosine is an important determinant of ischaemic (Zhao et al. 1993, 1994; Peart & Headrick, 2000) or hypoxic tolerance (Matherne et al. 1996). We previously acquired evidence that altered adenosine handling might contribute to impaired ischaemic tolerance with age (Headrick, 1998), and more recent evidence supports an age-related decline in adenosine-mediated protec