Daidzein (0.2 mg/Kg/s.c) (Sigma Aldrich [P] Ltd., Bangalore, India) was dissolved in dimethyl sulfoxide (DMSO) and then injected to the animals for 7 days, 3 weeks after the administration of streptozotocin. Sodium nitrite (10 μM/L) (Rankem, Fine Chemicals Ltd., New Delhi, India) and 5-Hydroxy Decanoate (100 μM/L) (Sigma Aldrich [P] Ltd., Bangalore, India) were added in minimum quantity of distilled water and added to Kreb's Henseleit solution. All other reagents used in this study were of analytical grade and always freshly prepared before use.

Total 12 groups have been used in present study each group consist of 6-10 Wistar rats (180-250) of either sex. Experimental diabetes was induced by single dose administration of streptozotocin (50 mg/kg, i.p) [³⁹]. There was 10% of mortality within 1^st week and 20% mortality was noted up to harvesting of heart. Serum glucose was estimated spectrophotometrically at 505 nm by glucose oxidase/pyruvate oxidase (GOD-POD) method [⁴⁰,⁴¹] using an enzymatic kit (Kamineni Life Sciences Pvt. Ltd. Hyderabad, India). Serum glucose level > 200 mg/dl were considered to be hyperglycaemic.

Rats were administered heparin (500 IU/L, i.p) 20 min. prior to sacrificing the animal by cervical dislocation. Heart was rapidly excised and immediately mounted on Langendorff's apparatus [⁴²]. Isolated heart was retrogradely perfused at constant pressure of 80 mmHg with Kreb's-Henseleit (KH) buffer (NaCl 118 mM; KCl 4.7 mM; CaCl2 2.5 mM; MgSO4.7H20 1.2 mM; KH2PO4 1.2 mM; C6H12O6 11 mM), pH 7.4, maintained at 37°C bubbled with 95% O2 and 5%CO2. Flow rate was maintained at 7-9 ml/min. using Hoffman's screw. The heart was enclosed in double wall jacket, the temperature of which was maintained by circulating water heated at 37°C. Ischemic preconditioning was produced by closing the inflow of K-H solution for 5 min followed by 5 min of reperfusion. Four such episodes were employed. Global ischemia was produced for 30 min. followed by 120 min. of reperfusion. Coronary effluent was collected before ischemia, immediately, 5 min. and 30 min. after reperfusion for estimation of Lactate Dehydrogenase (LDH) and Creatine Kinase (CK-MB) [⁴³].

The assessment of myocardial infarct size was done by using triphenyltetrazolium chloride (TTC) staining method, while LDH and CK-MB were estimated by using commercially available kits (LDH Siemens Medical Solution Diagnostics Ltd., Ajwa Road, Baroda, India, CK-MB Nicholas Piramal India Ltd., Mumbai). Values of LDH and CK-MB were expressed in international units per litre (IU/L).

The heart was removed from the Langendorff's apparatus. Both the atria and root of aorta were excised and ventricles were kept overnight at -4°C temperature. Frozen ventricles were sliced into uniform sections of about 1-2 mm thickness. The slices were incubated in 1% w/v triphenyltetrazolium chloride stain (TTC stain) at 37°C in 0.2 M Tris-chloride buffer for 30 min. The normal myocardium was stained brick red while the infarcted portion remained unstained. Infarct size was measured by the volume method [⁴⁴]

Nitrite is a stable nitrogen intermediate formed from the spontaneous degradation of NO. Unlike NO, nitrite can be measured easily and nitrite concentrations can be used to infer levels of NO production [^45-47]. Nitrite release in coronary effluent was measured [⁴⁸]. Greiss reagent 0.5 ml (1:1 solution of 1% sulphanilamide in 5% phosphoric acid and 0.1% N-(1-Naphthyl) ethylenediamine dihydrochloride in water) was added to 0.5 ml of coronary effluent. The optical density at 550 nm was measured using spectrophotometer (UV-1700 Spectrophotometer, Shimadzu, Japan). Nitrite concentration was calculated by comparison with spectrophotometer reading of standard solution of sodium nitrite prepared in K-H buffer [⁴⁸]

A diagrammatic representation of experimental protocol is shown in Figure 1. In all groups, isolated rat heart was perfused with K-H solution and allowed for 10 min of stabilization. Group 1 (Sham Control; n = 6): Isolated rat heart was perfused continuously for 200 min without subjecting them to global ischemia and reperfusion. Group 2 (Ischemia-Reperfusion Control; n = 6): After 10 min of stabilization, isolated rat heart preparation was subjected to 30 min. global ischemia followed by 120 min. of reperfusion. Group 3 (Ischemic Preconditioning Control; n = 6): After 10 min of stabilization, heart was subjected to four cycles of ischemic preconditioning, each cycle comprised of 5 min. global ischemia followed by 5 min. reperfusion further followed by 30 min. global ischemia and 120 min. of reperfusion. Group 4 (Ischemic Preconditioning in Diabetic Rats; n = 6): Isolated heart preparation from diabetic rat subjected to four cycles of ischemic preconditioning as described earlier in group 3. Group 5 (Ischemic Preconditioning in Daidzein (0.2 mg/Kg/s.c/day) Pre-treated Diabetic Rat; n = 6): Isolated rat heart preparation from daidzein (0.2 mg/Kg/s.c/day) pre-treated diabetic rat was subjected to four cycles of ischemic preconditioning as described earlier in group 3. Group 6 (Ischemic preconditioning in Sodium Nitrite (10 μM/L) perfused Normal Rat Heart; n = 6): After 10 min of stabilization, heart was perfused with K-H buffer containing sodium nitrite (10 μM/L) for 30 min. and then subjected to four cycles of ischemic preconditioning as described earlier in group 3. Group 7 (Ischemic preconditioning in Sodium Nitrite (10 μM/L) perfused Diabetic Rat Heart; n = 6): Isolated heart preparation obtained from diabetic rat was perfused with sodium nitrite (10 μM/L) for 30 min. followed by IPC as described in group 3. Group 8 (Ischemic Preconditioning in Daidzein (0.2 mg/Kg/s.c/day) Pre-treated, Sodium Nitrite (10 μM/L) Perfused Diabetic Rat Heart; n = 6): After 10 min of stabilization, Isolated rat heart preparation from daidzein (0.2 mg/Kg/s.c/day) pre-treated diabetic rat was perfused with sodium nitrite (10 μM/L) for 30 min. followed by as described in group 3. Group 9 (Ischemic Preconditioning in 5-Hydroxy Decanoate (100 μM/L) Perfused Normal Rat Heart; n = 6): After 10 min of stabilization, isolated heart was perfused with K-H buffer containing 5-hydroxy decanoate (100 μM/L) for 10 min. and then subjected to IPC as described earlier in group 3. Group 10 (Ischemic Preconditioning in Daidzein (0.2 mg/Kg/s.c/day) Pre-treated, 5-Hydroxy Decanoate (100 μM/L) Perfused Diabetic Rat Heart; n = 6): After 10 min of stabilization, isolated rat heart preparation from daidzein (0.2 mg/Kg/s.c/day) pre-treated diabetic rat perfused with 5-hydroxy decanoate (100 μM/L) containing K-H buffer for 10 min. followed by as described in group 3. Group 11 (Ischemic Preconditioning in 5-Hydroxy Decanoate (100 μM/L) and Sodium Nitrite (10 μM/L) Perfused Diabetic Rat Heart; n = 6): After 10 min of stabilization, isolated heart preparation from diabetic rat was perfused with K-H buffer containing 5-hydroxy decanoate (100 μM/L) for 10 min. which is further followed by perfusion with sodium nitrite (10 μM/L) for 30 min. followed by as described earlier in group 3. Group 12 (Ischemic Preconditioning in Daidzein (0.2 mg/Kg/s.c/day) Pre-treated, 5-Hydroxy Decanoate (100 μM/L) and Sodium Nitrite (10 μM/L) Perfused Diabetic Rat Heart; n = 6): After 10 min of stabilization, isolated rat heart preparation from daidzein (0.2 mg/Kg/s.c/day) treated diabetic rat was followedby group 11.

All values were expressed as mean ± standard deviation (S.D). Statistical analysis was performed using Sigmastat Software. Glucose value was compared by Student's paired t-test. The values of infarct size, LDH, CK-MB and nitrite level were statistically analysed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test as a post hoc test. Value of P < 0.05 was considered to be statistically significant.

The administration of single dose of streptozotocin (50 mg/Kg, i.p) significantly increased blood glucose as compared to basal value (Figure 2).

Global ischemia for 30 min followed by 120 min of reperfusion significantly increased the myocardial infarct size, as compared to sham control. Four episodes of IPC significantly decreased I/R induced increase in myocardial infarct size in normal rat heart. However, ischemic preconditioning failed to decrease the myocardial infarct size in diabetic rat heart. Moreover, IPC induced decrease of infarct size was significantly restored in DDZ pre-treated and in sodium nitrite perfused diabetic rat heart. However, perfusion with 5-HD significantly attenuated the decrease of myocardial infarct size in normal, DDZ pre-treated and sodium nitrite perfused diabetic rat heart alone or in combination (Figure 3).

Global ischemia for 30 min followed by 120 min of reperfusion markedly increased the release of LDH as compared to sham control. Four episodes of IPC significantly reduced the I/R induced increase in the release of LDH in normal rat heart but not in the diabetic rat heart. Moreover, IPC induced decrease in the release of LDH was significantly restored in DDZ pre-treated, and in sodium nitrite perfused diabetic rat heart. However, perfusion with 5-HD significantly attenuated the decrease in the release of LDH in normal, DDZ pre-treated and sodium nitrite perfused diabetic rat heart alone or in combination (Figure 4).

Global ischemia for 30 min followed by 120 min of reperfusion markedly increased the release of CK-MB, as compared to sham control. Four episodes of IPC significantly reduced the I/R induced increase in the release of CK-MB in normal rat heart but not in the diabetic rat heart. Moreover, IPC induced decrease release of CK-MB was significantly restored in DDZ pre-treated and sodium nitrite perfused diabetic rat heart. However, perfusion with 5-HD significantly attenuated the decrease in the release of CK-MB in normal, DDZ pre-treated and sodium nitrite perfused diabetic rat heart alone or in combination (Figure 5).

Four episodes of IPC significantly increased the release of nitrite into coronary effluent of normal animals, as compared to I/R group but not in isolated heart obtained from diabetic rat. Treatment with daidzein, a caveolin inhibitor (0.2 mg/Kg/s.c, one week), significantly increased, the release of nitrite in diabetic rat heart subjected to IPC (Figure 6).