Background-The mechanism of delayed preconditioning induced by activation of adenosine A 1 receptors (A 1 ARs) is not fully understood. We determined the role of inducible nitric oxide synthase (iNOS) in mediating adenosine-induced late cardioprotection using pharmacological inhibitors and iNOS gene-knockout mice. Methods and Results-Adult male mice were treated with saline or an A 1 AR agonist, 2-chloro-N 6 -cyclopentyladenosine (CCPA). Twenty-four hours later, the hearts were perfused in Langendorff mode and subjected to 30 minutes of global ischemia followed by 30 minutes of reperfusion. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.1 mg/kg IP) and S-methylisothiourea (SMT; 3 mg/kg IP) were used to block A 1 ARs and iNOS, respectively. Infarct size (IS) was measured by triphenyltetrazolium chloride staining, and iNOS expression was measured by Western blots. Myocardial IS was reduced from 24.0Ϯ3.2% in the saline group to 12.2Ϯ2.5% in CCPA-treated mice (PϽ0.05). The infarct-reducing effect of CCPA was abrogated by DPCPX (29.3Ϯ3.4%) and SMT (32.3Ϯ2.6%) and was absent in mice with targeted ablation of iNOS (23.9Ϯ1.6%). CCPA produced improvement in postischemic end-diastolic pressure, developed pressure, and rate-pressure product, which was also blocked by DPCPX and SMT. Increased iNOS protein expression observed in CCPA-treated hearts was diminished by DPCPX. Conclusions-Selective

Background-Heat-shock protein 70 (HSP 70) plays a role in myocardial protection. No studies are available, however, to show that direct gene transfer of HSP 70 reduces myocardial infarction in vivo. Methods and Results-Rabbit hearts were injected with vehicle or Ad.HSP70 at 3 sites (1.5ϫ10 9 pfu, 50 L/site) in the left ventricle (LV). Four days later, hearts were removed, and expression of inducible (HSP 70) and constitutive (HSC 70) proteins was measured in the LV and right ventricle (RV). Subsets of 5 to 7 animals in the vehicle-, Ad.lacZ-, and Ad.HSP70-treated groups were subjected to 30 minutes of ischemia and 3 hours of reperfusion. Infarct size was measured by tetrazolium staining. Increased expression of HSP 70 was observed in LV injected with Ad.HSP70 compared with vehicle-treated hearts. HSP 70 was undetectable in RV, the noninjected region of the heart. The expression of HSC 70 remained unchanged in hearts treated with vehicle or Ad.HSP70. Infarct size (% risk area) decreased to 24.5Ϯ2.8 in Ad.HSP70-injected hearts compared with 41.9Ϯ2.8 and 42.7Ϯ2.5 in the vehicle-and Ad.LacZ-treated hearts (PϽ0.01). The infarct size was not different between the vehicle-and Ad.LacZ-treated hearts (PϾ0.05). The risk areas (% of LV) were not different among the 3 groups, ie, 50.1Ϯ5.2, 47.7Ϯ3.5, and 53.3Ϯ2.9 in vehicle-, Ad.lacZ-, and Ad.HSP70-treated groups (PϾ0.05). Conclusions-Direct gene delivery of HSP 70 in vivo reduces the severity of ischemic injury in the heart. (Circulation.2001;103:877-881.)

Ischemic preconditioning (PC) induces delayed phase of protection, known as the second window of protection (SWOP). We investigated this phenomenon in rat and correlated it with the expression of 72-kDa heat shock protein (HSP 72). Rats were preconditioned with 1, 2, and 3 cycles of 5-min left anterior descending artery occlusions, each separated by a 10-min reperfusion (PC × 1, PC × 2 and PC × 3, respectively). Another group of rats was preconditioned with heat shock (HS) by raising temperature to 42°C for 15 min. Twenty-four hours later, rats were given sustained ischemia for 30 min and 90 min of reperfusion. Infarct sizes (%risk area) were 40.0 ± 7.5, 37.6 ± 5.6, and 47.6 ± 2.4 (mean ± SE) for PC × 1, PC × 2, and PC × 3 hearts, respectively, which were not different from the sham (49.9 ± 3.9, P > 0.05). In contrast, infarct size was reduced from 47.5 ± 3.8% in sham to 4.7 ± 2.3% ( P < 0.01) 24 h after HS. Additionally, early PC significantly reduced infarct size from 47.5 ± 3.8% in controls to 6.0 ± 1.2 and 5.0 ± 1.1% with PC × 1 and PC × 3. Repeated PC cycles induced over a threefold increase in HSP 70 mRNA after 2 h compared with sham ( P < 0.05). HSP 72, which increased 24 h after PC or HS, was not significantly different between the two PC stimuli. We conclude that PC does not induce SWOP in rat heart despite enhanced expression of HSP 72. In contrast, HS-induced delayed protection was associated with enhanced accumulation of HSP 72. It is possible that SWOP and HS have distinct mechanisms of protection that may not be exclusively related to HSP 72 expression.

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