Respiratory constraints during activities in daily life and the impact on health status in patients with early-stage COPD: a cross-sectional study.

In patients with chronic obstructive pulmonary disease (COPD), exercise capacity is reduced, resulting over time in physical inactivity and worsened health status. It is unknown whether ventilatory constraints occur during activities of daily life (ADL) in early stages of COPD. The aim of this study was to assess respiratory mechanics during ADL and to study its consequences on dyspnoea, physical activity and health status in early-stage COPD compared with healthy controls. In this cross-sectional study, 39 early-stage COPD patients (mean FEV1 88±s.d. 12% predicted) and 20 controls performed 3 ADL: climbing stairs, vacuum cleaning and displacing groceries in a cupboard. Respiratory mechanics were measured during ADL. Physical activity was measured with accelerometry. Health status was assessed by the Nijmegen Clinical Screening Instrument. Compared with controls, COPD patients had greater ventilatory inefficiency and higher ventilatory requirements during ADL (P<0.05). Dyspnoea scores were increased in COPD compared with controls (P<0.001). During ADL, >50% of the patients developed dynamic hyperinflation in contrast to 10-35% of the controls. Higher dyspnoea was scored by patients with dynamic hyperinflation. Physical activity was low but comparable between both groups. From the patients, 55-84% experienced mild-to-severe problems in health status compared with 5-25% of the controls. Significant ventilatory constraints already occur in early-stage COPD patients during common ADL and result in increased dyspnoea. Physical activity level is not yet reduced, but many patients already experience limitations in health status. These findings reinforce the importance of early diagnosis of COPD and assessment of more than just spirometry.

[Medicinal intervention for COPD; even in patients with mild or moderate COPD?]. / Medicamenteuze interventie bij COPD, ook bij patiënten met lichte en matige COPD?

The classification of COPD based only on the presence of airway obstruction fails to provide insight into the burden of the disease, quality of life and prognosis. The severity of symptoms, degree of exercise intolerance and presence of comorbidity are also determinants for classifying the severity of the disease. COPD starts with abnormalities in the bronchiolar compartment which cause obstruction in the airways. This results in incomplete expiration; first during exercise and later, also at rest. This is called dynamic hyperinflation or air trapping. Such changes in the mechanics of breathing occur early in course of the disease, even in mild COPD (the GOLD I stage), and contribute to physical inactivity and deconditioning. Maximal bronchodilation--more precisely: bronchiolodilation--reduces the mechanism of dynamic hyperinflation inasmuch as the condition allows. This has a positive effect on the symptoms of dyspnoea during exercise and thus on exercise capacity and trainability, even early on in the disease. Medicinal therapy has a positive effect on the progression of COPD, also in the early stages of disease.

Diagnostic accuracy of metronome-paced tachypnea to detect dynamic hyperinflation.

INTRODUCTION: This prospective study was carried out to investigate if metronome-paced tachypnea (MPT) can serve as an accurate diagnostic tool to identify patients with chronic obstructive pulmonary disease (COPD) who are susceptible to develop dynamic hyperinflation during exercise. Commonly, this is assessed by measuring change in inspiratory capacity (IC) during cardiopulmonary exercise testing (CPET), which, however, is complex and laborious. METHODS: Fifty-three patients with COPD (FEV(1) 58 ± 22%pred) and 20 age-matched healthy subjects were characterized by lung function testing and performed CPET (reference standard) and MPT. The repeatability coefficient of IC (10·2%) was used as cut-off to classify subjects as hyperinflators during CPET. Subsequently, dynamic hyperinflation was measured after MPT. With receiver operating characteristic analysis, the optimal cut-off for MPT-induced dynamic hyperinflation was determined and sensitivity and specificity of MPT to identify hyperinflators were evaluated. RESULTS: With 10·2% decrease in IC as cut-off for CPET-induced dynamic hyperinflation, the optimal cut-off for MPT was 11·1% decrease in IC. Using these cut-offs, MPT had a sensitivity of 85% and specificity of 85% to identify the subjects who hyperinflated during CPET. CONCLUSIONS: The MPT test shows good overall accuracy to identify subjects who are susceptible to develop dynamic hyperinflation during CPET. Before considering the use of MPT as a screening tool for dynamic hyperinflation in COPD, sensitivity and specificity need further evaluation.

COPD Anno 2011: emphasis on bronch(iol)odilation.

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of disability and death worldwide. Although COPD is considered to be a preventable and treatable disease, there are concerns that COPD remains substantially underdiagnosed and undertreated. Even in mild disease, patients suffer from significant impairments in health status, which places a considerable burden on patients as well as society. Symptomatic patients are likely to progress to more advanced disease. To avoid breathlessness, they adapt and gradually reduce their activities, which, inevitably, leads to further deconditioning. As a consequence, a progressive deterioration in physical activity with increasing severity of COPD can be observed. Because physical activity is closely related to exacerbation rate, hospitalization, and mortality in patients with COPD, it is important to recognize the role of pharmaceutical interventions in enabling patients to stay physically active. Bronch(iol)odilation not only has important direct effects (symptom relief), but also exerts indirect effects on exercise capacity, exacerbation rate, health status, and mortality. In patients with COPD, the latter effects may be even more important than the direct effects. In this review the current view on causes and consequences of activity limitation in COPD is summarized. From this perspective, the rationale behind bronch(iol)odilator therapy as the cornerstone of treatment for patients with COPD will be discussed.

Impact of wall thickness on conduit artery function in humans: is there a "Folkow" effect?

Regional heterogeneity in wall architecture and thickness may be present between conduit arteries in the upper and lower limbs in humans. These differences in wall architecture may, in turn, influence vascular responsiveness. Folkow proposed in the 1950s that heterogeneity in wall-to-lumen ratio (W:L) could contribute to differences in vascular responsiveness, but this hypothesis has never been directly confirmed in vivo. Our first aim was to examine wall thickness and W:L across arteries in the lower (common and superficial femoral) and upper limbs (brachial and radial) of healthy men (n=35) using high resolution ultrasound. In a subgroup (n=20) we examined the relationship between W:L of these arteries, physiological (flow-mediated dilation, FMD) and pharmacological vasodilation (glyceryl trinitrate, GTN). Diameter and wall thickness differed significantly across all arteries (ANOVA P<0.001), with smaller arteries having a relatively larger wall thickness. Moreover, we found a significant correlation between W:L and the FMD-response (r=0.55, P<0.001), which remained significant after correcting for the eliciting shear stress (r=0.47, P<0.001), indicating that W:L/FMD relationship was not primarily related to the impact of diameter on the shear rate stimulus to FMD. W:L also correlated strongly with the GTN-response (r=0.56, P<0.001) across all arteries studied. These results indicate that regional heterogeneity exists in W:L within, but also between, limbs. More importantly, differences in W:L contribute to differences in vascular functional responses, reinforcing the conceptual proposal of Folkow, who suggested that arteries with larger W:L exhibit exaggerated responses to vasoactive stimuli.

Endogenous adenosine selectively modulates oxidant stress via the A1 receptor in ischemic hearts.

We tested the impact of A1 adenosine receptor (AR) deletion on injury and oxidant damage in mouse hearts subjected to 25-min ischemia/45-min reperfusion (I/R). Wild-type hearts recovered approximately 50% of contractile function and released 8.2 +/- 0.7 IU/g of lactate dehydrogenase (LDH). A1AR deletion worsened dysfunction and LDH efflux (15.2 +/- 2.6 IU/g). Tissue cholesterol and native cholesteryl esters were unchanged, whereas cholesteryl ester-derived lipid hydroperoxides and hydroxides (CE-O(O)H; a marker of lipid oxidation) increased threefold, and alpha-tocopherylquinone [alpha-TQ; oxidation product of alpha-tocopherol (alpha-TOH)] increased sixfold. Elevations in alpha-TQ were augmented by two- to threefold by A1AR deletion, whereas CE-O(O)H was unaltered. A(1)AR deletion also decreased glutathione redox status ([GSH]/[GSSG + GSH]) and enhanced expression of the antioxidant response element heme oxygenase-1 (HO-1) during I/R: fourfold elevations in HO-1 mRNA and activity were doubled by A1AR deletion. Broad-spectrum AR agonism (10 microM 2-chloroadenosine; 2-CAD) countered effects of A1AR deletion on oxidant damage, HO-1, and tissue injury, indicating that additional ARs (A(2A), A(2B), and/or A3) can mediate similar actions. These data reveal that local adenosine engages A1ARs during I/R to limit oxidant damage and enhance outcome selectively. Control of alpha-TOH/alpha-TQ levels may contribute to A1AR-dependent cardioprotection.

Cardiac and coronary function in the Langendorff-perfused mouse heart model.

The Langendorff mouse heart model is widely employed in studies of myocardial function and responses to injury (e.g. ischaemia). Nonetheless, marked variability exists in its preparation and functional properties. We examined the impact of early growth (8, 16, 20 and 24 weeks), sex, perfusion fluid [Ca(2+)] and pacing rate on contractile function and responses to 20 min ischaemia followed by 45 min reperfusion. We also assessed the impact of strain, and tested the utility of the model in studying coronary function. Under normoxic conditions, hearts from 8-week-old male C57BL/6 mice (2 mm free perfusate [Ca(2+)], 420 beats min(-1)) exhibited 145 +/- 2 mmHg left ventricular developed pressure (LVDP). Force development declined by approximately 15% (126 +/- 5 mmHg) with a reduction in free [Ca(2+)] to 1.35 mm, and by 25% (108 +/- 3 mmHg) with increased pacing to 600 beats min(-1). While elevated heart rate failed to modify ischaemic outcome, the lower [Ca(2+)] significantly improved contractile recovery (by >30%). We detected minimal sex-dependent differences in normoxic function between 8 and 24 weeks, although age modified contractile function in males (increased LVDP at 24 versus 8 weeks) but not females. Both male and female hearts exhibited age-related reductions in ischaemic tolerance, with a significant decline in recovery evident at 16 weeks in males and later, at 20-24 weeks, in females (versus recoveries in hearts at 8 weeks). Strain also modified tolerance to ischaemia, with similar responses in hearts from C57BL/6, 129/sv, Quackenbush Swiss and FVBN mice, but substantially greater tolerance in BALB/c hearts. In terms of vascular function, baseline coronary flow (20-25 ml min(-1) g(-1)) was 50-60% of maximally dilated flows, and coronary reactive and functional hyperaemic responses were pronounced (up to 4-fold elevations in flow in hearts lacking ventricular balloons). These data indicate that attention to age (and sex) of mice will reduce variability in contractile function and ischaemic responses. Even small differences in perfusion fluid [Ca(2+)] also significantly modify tolerance to ischaemia (whereas modest shifts in heart rate do not impact). Ischaemic responses are additionally strain dependent, with BALB/c hearts displaying greatest intrinsic tolerance. Finally, the model is applicable to the study of vascular reactivity, providing large responses and excellent reproducibility.

Modulation of ischaemic contracture in mouse hearts: a 'supraphysiological' response to adenosine.

While inhibition of ischaemic contracture was one of the first documented cardioprotective actions of exogenously applied adenosine, it is not known whether this is a normal function of endogenous adenosine generated during ischaemic stress. Additionally, the relevance of delayed contracture to postischaemic outcome is unclear. We tested the ability of endogenous versus exogenous adenosine to modify contracture (and postischaemic outcomes) in C57/Bl6 mouse hearts. During ischaemia, untreated hearts developed peak contracture (PC) of 85 +/- 5 mmHg at 8.9 +/- 0.8 min, with time to reach 20 mmHg (time to onset of contracture; TOC) of 4.4 +/- 0.3 min. Adenosine (50 microm) delayed TOC to 6.7 +/- 0.6 min, as did pretreatment with 10 microm 2-chloroadenosine (7.2 +/- 0.5 min) or 50 nm of A(1) adenosine receptor (AR) agonist N(6)-cyclohexyladenosine (CHA) (6.7 +/- 0.3 min), but not A(2A)AR or A(3)AR agonists (20 nm 2-[4-(2-carboxyethyl) phenethylamino]-5' N-methylcarboxamidoadenosine (CGS21680) or 150 nm 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA), respectively). Adenosinergic contracture inhibition was eliminated by A(1)AR gene knockout (KO), mimicked by A(1)AR overexpression, and was associated with preservation of myocardial [ATP]. This adenosine-mediated inhibition of contracture was, however, only evident after prolonged (10 or 15 min) and not brief (3 min) pretreatment. Ischaemic contracture was also insensitive to endogenously generated adenosine, since A(1)AR KO, and non-selective and A(1)AR-selective antagonists (50 microm 8-sulphophenyltheophylline and 150 nm 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX), respectively), all failed to alter intrinsic contracture development. Finally, delayed contracture with A(1)AR agonism/overexpression or ischaemic 2,3-butanedione monoxime (BDM; 5 microm to target Ca(2+) cross-bridge formation) was linked to enhanced postischaemic outcomes. In summary, adenosinergic inhibition of contracture is solely A(1)AR mediated; the response is 'supraphysiological', evident only with significant periods of pre-ischaemic AR agonism (or increased A(1)AR density); and ischaemic contracture appears insensitive to locally generated adenosine, potentially owing to the rapidity of contracture development versus the finite time necessary for expression of AR-mediated cardioprotection.

Contractile effects of adenosine, coronary flow and perfusion pressure in murine myocardium.

There is mixed evidence adenosine receptors (ARs) may enhance myocardial contractility, although this remains contentious. We assessed inotropic actions of adenosine (50 muM) and selective AR activation with 100 nM N (6)-cyclohexyladenosine (CHA; A(1)AR agonist), 25 nM 2-[p-(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS-21680; A(2A)AR agonist) and 100 nM 2-chloro-N (6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA; A(3)AR agonist) in mouse hearts perfused at constant pressure, constant flow, or conditions of stable flow and pressure (following maximal nitroprusside-mediated dilatation at constant flow). Adenosine and CGS-21680 significantly (although modestly) increased force in constant-pressure perfused hearts (

Effects of adenosine deaminase and A1 receptor deficiency in normoxic and ischaemic mouse hearts.

OBJECTIVE: Adenosine deaminase (ADA) may be multifunctional, regulating adenosine levels and adenosine receptor (AR) agonism, and potentially modifying AR functionality. Herein we assess effects of ADA (and A1AR) deficiency on AR-mediated responses and ischaemic tolerance. METHODS: Normoxic function and responses to 20 or 25 min ischaemia and 45 min reperfusion were studied in isolated hearts from wild-type mice and from mice deficient in ADA and/or A1ARs. RESULTS: Neither ADA or A1AR deficiency significantly modified basal contractility, although ADA deficiency reduced resting heart rate (an effect abrogated by A1AR deficiency). Bradycardia and vasodilation in response to AR agonism (2-chloroadenosine) were unaltered by ADA deficiency, while A1AR deficiency eliminated the heart rate response. Adenosine efflux increased 10- to 20-fold with ADA deficiency (at the expense of inosine). Deletion of ADA improved outcome from 25 min ischaemia, reducing ventricular diastolic pressure (by 45%; 21+/-4 vs. 38+/-3mm Hg) and lactate dehydrogenase (LDH) efflux (by 40%; 0.12+/-0.01 vs. 0.21+/-0.02 U/g/min ischaemia), and enhancing pressure development (by 35%; 89+/-6 vs. 66+/-5mm Hg). Similar protection was evident after 20 min ischaemia, and was mimicked by the ADA inhibitor EHNA (5 microM). Deletion of ADA also enhanced tolerance in A1AR deficient hearts, though effects on diastolic pressure were eliminated. CONCLUSIONS: Deficiency of ADA does not alter sensitivities of cardiovascular A1 or A2ARs (despite markedly elevated [adenosine]), but significantly improves ischaemic tolerance. Conversely, A1AR deficiency impairs ischaemic tolerance. Effects of ADA deficiency on diastolic pressure appear solely A1AR-dependent while other ARs or processes additionally contribute to improved contractile recovery and reduced cell death.

Age-associated shifts in cardiac gene transcription and transcriptional responses to ischemic stress.

Aged hearts exhibit reduced tolerance to ischemia-reperfusion, together with altered structure and post-ischemic remodelling. The molecular bases of such changes are unclear. Using cDNA microarrays and quantitative RT-PCR we characterized shifts in gene expression patterns with aging in normoxic and post-ischemic (20 min global ischemia, 60 min reperfusion) murine hearts (young: 2-4 months; aged: 16-18 months). We identified an age-associated up-regulation of transcripts involved in cell death, oxygen transport and metabolism in normoxic hearts. Down-regulated transcripts were involved in transporter activity, protein binding and hydrolase activity, changes in MAPK, WNT and TGF-beta signalling with aging were also observed. Ischemic stress generated a much greater degree of contractile impairment and cellular damage in aged vs. young hearts. This was associated with a substantially modified transcriptional response, with selective changes in Ca2+, WNT, NOTCH and G-protein coupled receptor signalling paths in aged vs. young hearts. Despite some common responses to ischemia in young and aged hearts (induction of heat shock protein transcripts), aging selectively modified ischemic responses of immediate early genes, and genes involved in modulating apoptosis and remodelling/angiogenesis. In summary, aging is associated with shifts in cardiovascular gene expression consistent with the phenotypic features of older hearts. Reduced tolerance with age may be related to modification of signalling (particularly WNT and TGF-beta), and shifts in expression of immediate early genes, and genes important in control of cell death/survival, angiogenesis, and cardiac remodelling.

Adenosine-mediated cardioprotection in the aging myocardium.

With aging, it appears the heart's ability to withstand injury declines markedly. Unfortunately, the incidence of ischemic disorders increases dramatically with age. Though the genesis of the ischemia-intolerant phenotype is incompletely understood (and likely multi-factorial), it may involve changes in intrinsic cardioprotective responses. In this respect we and others have interrogated the role of the adenosine receptor (AR) system in dictating ischemic tolerance and the impact of age on AR-mediated cardioprotection. It is intriguing to note ARs impact on many processes implicated in myocardial 'aging': adenosine counters Ca2+ influx and oxidant injury, modifies substrate metabolism to improve tolerance, is pro-angiogenic, inhibits myocardial fibrosis, and can limit apoptosis. Thus, dysregulation of the AR system could contribute to many features of aged hearts (including ischemic intolerance). The latter is borne out by observations that AR-mediated protective responses decline with intrinsic tolerance and that transgenic manipulation of the AR system restores intrinsic tolerance and protective responses in aged hearts. Mechanisms underlying failure in adenosinergic protection remain undefined. Here we review data on the effects of aging on cardiovascular AR transcription and expression, generation of signal (adenosine formation), and protective signaling coupled to ARs.

Protecting murine hearts from ischaemia-reperfusion using selective inhibitors of adenosine metabolism.

1. By selectively modifying adenosine metabolism via adenosine deaminase or adenosine kinase inhibitors, it may be possible to enhance the receptor-mediated protective actions of adenosine in a site- and event-specific fashion. 2. We characterized cardioprotective actions of the adenosine deaminase inhibitor erythro-2-(2-hydroxy-3-non-yl)adenine (EHNA) and the adenosine kinase inhibitor iodotubercidin in C57/Bl6 mouse hearts subjected to 20 min global normothermic ischaemia and 40 min reperfusion. 3. Ventricular pressure development only recovered to 45 +/- 2% of baseline levels (67 +/- 5 mmHg) in untreated hearts, with sustained and pronounced diastolic contracture (25 +/- 2 mmHg). Treatment with 20 micromol/L EHNA increased recovery of ventricular pressure (107 +/- 9 mmHg), reduced postischaemic diastolic pressure (13 +/- 1 mmHg) and reduced loss of lactate dehydrogenase (LDH; an indicator of necrotic damage) by 50% (9 +/- 2 vs 19 +/- 2 IU/g). Adenosine kinase inhibition with 10 micromol/L iodotubercidin also improved pressure development (to 100 +/- 8 mmHg) and reduced LDH efflux (5 +/- 2 IU/g). 4. Protective actions were mimicked by adenosine and inhibited by adenosine receptor antagonism (50 micromol/L 8-rho-sulfophenyltheophylline) and mitochondrial K(ATP) channel inhibition (50 micromol/L 5-hydroxydecanoate). 5. Coinfusion of the inhibitors, 'trapping' formed adenosine, failed to exert protection and, in some instances, was detrimental. Although substantial benefit was gained by these agents in hearts from young animals, neither inhibitor was effective in 'aged' hearts (18 months). 6. Our data demonstrate that adenosine deaminase or kinase inhibition substantially limits injury during ischaemia-reperfusion. Protection involves adenosine receptor activation. However, cardioprotection via either enzyme inhibitor requires an alternative purine-salvage pathway to be functional and was reduced in aged hearts known to be increasingly susceptible to ischaemic damage.

Age-related changes in ischemic tolerance in male and female mouse hearts.

Aging is associated with reduced tolerance to ischemic insult, and genesis of this intolerant phenotype is poorly understood. We characterized effects of aging and gender on cardiovascular function and cell damage during 20 min ischemia and 60 min reperfusion in isolated hearts from young adult (2-4 months), mature adult (8 months), middle-aged (12 months), aged (18 months), and senescent (24-28 months) C57/Bl6 mice. Aging substantially impaired recovery of ventricular contractility, with this change primarily evident within 12 months of age. In males ventricular developed pressure recovered to 72 +/- 8 mmHg in young hearts vs. only 44 +/- 7, 30 +/- 3, 24 +/- 5, and 27 +/- 4 mmHg in mature, middle-aged, aged and senescent hearts, respectively. This pattern was largely due to worsened diastolic dysfunction. Coronary flow recovered to below pre-ischemic levels in all ages, correlating with contractile recovery. However, coronary dysfunction (impaired responses to 2-chloroadenosine and ADP) was unaltered by senescence. Lactate dehydrogenase (LDH) loss, a marker for oncosis, increased to middle-age (approximately twofold), then fell with further aging to a value no longer different from that in young adult hearts. Similar patterns of change were observed in female hearts, although LDH efflux was significantly lower in mature adult and middle-aged female vs. male hearts, with functional tolerance also tending to be greater at these ages (though not achieving significance). Overall, our data reveal age-related ischemic intolerance develops well before senescence, being primarily evident by "middle-age". Phenotypic changes appear selective for myocardial vs. vascular injury, and functional vs. oncotic injury. Similar changes occur in males and females, though there is evidence of a protected phenotype in mature to middle-aged female vs. male hearts.

Genetic deletion of the A1 adenosine receptor limits myocardial ischemic tolerance.

Adenosine receptors may be important determinants of intrinsic ischemic tolerance. Genetically modified mice were used to examine effects of global A1 adenosine receptor (A1AR) knockout (KO) on function and ischemic tolerance in perfused mouse hearts. Baseline contractile function and heart rate were unaltered by A1AR KO, which was shown to abolish the negative chronotropic effects of 2-chloroadenosine (A1AR-mediated) without altering A2 adenosine receptor-mediated coronary dilation. Tolerance to 25 minutes global normothermic ischemia (followed by 45 minutes reperfusion) was significantly limited by A1AR KO, with impaired contractile recovery (reduced by 25%) and enhanced lactate dehydrogenase (LDH) efflux (increased by 100%). Functional effects of A1AR KO involved worsened systolic pressure development with little to no change in diastolic dysfunction. In contrast, cardiac specific A1AR overexpression enhanced ischemic tolerance with a primary action on diastolic dysfunction. Nonselective receptor agonism (10 micromol/L 2-chloroadenosine) protected wild-type and also A1AR KO hearts (albeit to a lesser extent), implicating protection via subtypes additional to A1ARs. However, A1AR KO abrogated effects of 2-chloroadenosine on ischemic contracture and diastolic dysfunction. These data are the first demonstrating global deletion of the A1AR limits intrinsic myocardial resistance to ischemia. Data indicate the function of intrinsically activated A1ARs appears primarily to be enhancement of postischemic contractility and limitation of cell death.

Effects of aging and ischemia on adenosine receptor transcription in mouse myocardium.

The well-documented age-related change in ischemic tolerance may result from impaired adenosine-mediated cardioprotection. Additionally, ischemia itself may potentially modify adenosine signalling, contributing to the post-ischemic phenotype. This study investigates age- and ischemia-dependent changes in adenosine receptor transcript levels (Adora) for the A(1), A(2A), A(2B), and A(3) receptor subtypes in mouse myocardium. Hearts from young (2-4 months) and moderately aged (16-18 months) mice were subjected to 20-min ischemia and 45-min reperfusion. Ischemic tolerance was impaired in aged hearts, which recovered less than 30% ventricular pressure development (compared with approximately 70% in young hearts), and lost 2-fold higher levels of lactate dehydrogenase during reperfusion (reflecting cellular disruption). Real-time PCR analyses revealed an age-related decline in Adora3 levels and induction of Adora2B. Curiously, this effect was mimicked by ischemia, which acutely reduced Adora3 levels and induced Adora2B in young (but not old) hearts. In contrast, in aged hearts ischemia selectively reduced levels of Adora1 transcript ( approximately 2-fold) without altering transcript levels for the other receptors. These results demonstrate selective modulation of cardioprotective adenosine receptor transcription by both aging and ischemia. Reduced A(3) adenosine receptor transcription may contribute to impaired ischemic tolerance in aged hearts, whereas changes in Adora transcription induced by ischemia may impact on the post-ischemic phenotype at later time points.

Aging-related changes in myocardial purine metabolism and ischemic tolerance.

Impaired tolerance to ischemia-reperfusion in older hearts may stem in part from alterations in purine catabolism, impacting on maintenance of energy state and protective signaling via extracellular adenosine. We characterized effects of aging on normoxic and post-ischemic purine metabolism in hearts from young (2-4 month), middle-aged (12 month), old (18 month), and senescent (24-28 month) C57/Bl6 mice. Normoxic function was similar in all age groups while normoxic purine efflux increased gradually with age. This was the result of enhanced efflux of hypoxanthine, xanthine and uric acid, with extracellular accumulation of adenosine and inosine remaining unchanged. While total purine washout during 60 min reperfusion following 20 min global ischemia was unaltered by aging (1057+/-109 nmoles/g in young vs. 1221+/-127 nmoles/g in senescent hearts), selective changes in purine catabolism were evident. Accumulation of adenosine and inosine were reduced by 50 and 80%, respectively, matched by 400 and 300% elevations in hypoxanthine and xanthine accumulation, respectively. Uric acid remained unchanged. Thus, while adenosine and inosine represented 15+/-2 and 47+/-3% of total purine efflux in young hearts, these values decreased to only 6+/-1 and 9+/-2% in senescent hearts. Efflux of IMP also increased 500% with aging whereas 5'-AMP was unaltered. These changes were associated with a substantial fall in ischemic tolerance, with left ventricular developed pressure recovering to 46+/-3% in young hearts vs. only 24+/-6, 16+/-4, and 19+/-4% in middle-age, old and senescent hearts, respectively. Our data collectively support a pronounced shift in purine catabolism, with reduced accumulation of salvageable and cardioprotective adenosine, and enhanced accumulation of poorly salvaged (and potentially injurious) hypoxanthine and xanthine. Mechanisms underlying this shift have yet to be determined. However, this may play a role in the marked decline in myocardial tolerance to ischemia with aging and senescence.

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

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 (2-4 months) and moderately aged (16-18 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 g-1 in young) whereas the incidence of tachyarrhythmias was similar in young (15 +/- 1 %) and aged hearts (16 +/- 1 %). Functional analysis confirmed equipotent effects of 50 micro M adenosine at A1 and A2 receptors in young and aged hearts. Nonetheless, while 50 micro M adenosine improved diastolic (5 +/- 1 mmHg) and developed pressures (134 +/- 7 mmHg) and LDH loss (6 +/- 2 IU g-1) 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 micro M diazoxide reduced ischaemic damage and arrhythmogenesis for both ages. Contractile and anti-necrotic effects of adenosine were limited by 100 micro M 5-hydroxydecanoate (5-HD) and 3 micro M chelerythrine. Anti-arrhythmic effects were limited by 5-HD but not chelerythrine. Non-selective (100 micro M 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 mg-1 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.

Receptor and non-receptor-dependent mechanisms of cardioprotection with adenosine.

The relative roles of mitochondrial (mito) ATP-sensitive K(+) (mitoK(ATP)) channels, protein kinase C (PKC), and adenosine kinase (AK) in adenosine-mediated protection were assessed in Langendorff-perfused mouse hearts subjected to 20-min ischemia and 45-min reperfusion. Control hearts recovered 72 +/- 3 mmHg of ventricular pressure (50% preischemia) and released 23 +/- 2 IU/g lactate dehydrogenase (LDH). Adenosine (50 microM) during ischemia-reperfusion improved recovery (149 +/- 8 mmHg) and reduced LDH efflux (5 +/- 1 IU/g). Treatment during ischemia alone was less effective. Treatment with 50 microM diazoxide (mitoK(ATP) opener) during ischemia and reperfusion enhanced recovery and was equally effective during ischemia alone. A(3) agonism [100 nM 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide], A(1) agonism (N(6)-cyclohexyladenosine), and AK inhibition (10 microM iodotubercidin) all reduced necrosis to the same extent as adenosine, but less effectively reduced contractile dysfunction. These responses were abolished by 100 microM 5-hydroxydecanoate (5-HD, mitoK(ATP) channel blocker) or 3 microM chelerythrine (PKC inhibitor). However, the protective effects of adenosine during ischemia-reperfusion were resistant to 5-HD and chelerythrine and only abolished when inhibitors were coinfused with iodotubercidin. Data indicate adenosine-mediated protection via A(1)/A(3) adenosine receptors is mitoK(ATP) channel and PKC dependent, with evidence for a downstream location of PKC. Adenosine provides additional and substantial protection via phosphorylation to 5'-AMP, primarily during reperfusion.

Coronary function and adenosine receptor-mediated responses in ischemic-reperfused mouse heart.

OBJECTIVES: To assess the impact of ischemia-reperfusion (I/R) on coronary function, and the role of endogenous adenosine in modifying post-ischemic vascular function in asanguinous hearts. METHODS: Vascular function was studied in Langendorff perfused mouse hearts subjected to 20-25-min ischemia and 30-min reperfusion. RESULTS: Ischemia altered the dependence of flow on work-rate observed in normoxic hearts, and inhibited reflow by mechanisms additional to diastolic compression. Coronary responses were selectively reduced: 2-chloroadenosine and ADP dilated with pEC(50)s of 8.4+/-0.1 and 7.4+/-0.1 in non-ischemic hearts versus 7.7+/-0.1 and 7.1+/-0.1 after 20-min ischemia (P<0.05). Sensitivity was further reduced after 25-min ischemia. Responses to nitroprusside were unaltered. NO-synthase antagonism (50 microM nitro-L-arginine methylester) reduced sensitivities to 2-chloroadenosine and ADP up to 10-fold, and eliminated inhibitory effects of I/R. K(ATP) blockade with 5 microM glibenclamide impaired sensitivity pre- and post-ischemia, not eliminating the inhibitory effects of I/R. A(1) adenosine receptor antagonism with 100 nM 8-cyclopentyl-1,3-dipropylxanthine worsened effects of ischemia on sensitivity. A(2A) adenosine receptor antagonism with 100 nM 8-(3-chlorostyryl)caffeine reduced post-ischemic flow by 50%, yet paradoxically enhanced post-ischemic contractile recovery. CONCLUSIONS: Ischemia modifies vascular control and impairs NO- versus K(ATP)-dependent coronary dilation in an asanguinous model. Endogenous adenosine protects against vascular dysfunction via A(1) receptors, and determines coronary reflow via A(2A) receptors. However, intrinsic A(2A) activation apparently worsens contractile dysfunction.