Cardioprotecive Properties of Known Agents in Rat Ischemia-Reperfusion Model Under Clinically Relevant Conditions: Only the NAD Precursor Nicotinamide Riboside Reduces Infarct Size in Presence of Fentanyl, Midazolam and Cangrelor, but Not Propofol

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  Figure 1. Experimental protocols. Following surgery and 3 min drug infusion, rat hearts were subjected to 30 min stabilization, 25 min ischemia (I) and 120 min reperfusion (R). Blood samples were collected before surgery (0.15 ml), 5 min before ischemia (0.15 ml) and 30 min after reperfusion (0.15 + 1 ml); (A) Protocol 1: For examining dose-responses of Empagliflozin, Fingolimod, nicotinamide riboside (NR) and Melatonin, rats were randomly assigned to 12 groups: saline, 10, 25, or 125 mg/kg empagliflozin, 1, 3, or 10 mg/kg Fingolimod, 100, 500, or 1,000 μg/kg Melatonin, 50 or 150 mg/kg NR. (B) Protocol 2: For examining NR (50 mg/kg, dissolved in saline) and Melatonin (500 μg/kg, dissolved in 0.99% ethanol) effects on in vivo IR injury, rats were randomly assigned to 5 groups: saline, saline + ethanol (0.99%), melatonin, NR, and NR + melatonin. (C) Protocol 3: For examining NR effects on in vivo IR injury in presence of cangrelor. Rats were randomly treated with saline or NR (50 mg/kg). Cangrelor (60 μg/kg) was infused at 10 min before reperfusion and maintained (360 μg/kg/h) until the end of reperfusion; (D) Protocol 4: For examining NR (50 mg/kg) effects on in vivo IR injury in presence…

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  Figure 2. Pilot dose-response of potential cardioprotective agents. The experiment followed protocol 1, rats were randomly subjected to empagliflozin [Empa;10 (n = 4), 25 (n = 4), 125 (n = 5) mg/kg], fingolimod [1 (n = 4), 3 (n = 4), 10 (n = 4) mg/kg], melatonin [100 (n = 4), 500 (n = 6), 1,000 (n = 4) μg/kg], or nicotinamide riboside [NR; 50 (n = 5), 150 (n = 5) mg/kg] or saline only (control; n = 8). Infarct size (IS, %) was related to area at risk (AAR). Data was analyzed by Kruskal-Wallis test with Dunnett post-hoc test (saline group as control category).

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  Figure 3. NR treatment alone showed maximal protection against in vivo cardiac IR injury. NR (50 mg/kg) treatment (n = 8) alone before ischemic insult reduced infarction, but not melatonin (500 μg/kg; n = 7) or NR + melatonin (n = 7). (A) Area at risk (AAR) related to left ventricle area (LV); (B) Image of TTC staining; (C) Infarct size related to AAR. (D) LDH release. *P < 0.05, **P < 0.01 vs. Saline group (n = 7), data was analyzed by one-way ANOVA with Bonferroni post hoc test.

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  Figure 4. Blood glucose level from the 5 experimental groups at three time points. Glucose (mmol/L) levels were detected before surgery, at 25 min baseline and 30 min reperfusion. **P < 0.01 vs. T1, data was analyzed using two-way Repeated Measures ANOVA applying the Bonferroni multiple comparisons test when relevant. All groups n = 6–8 experiments.

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  Figure 5. NR effects against in vivo cardiac IR injury in presence of cangrelor. NR treatment reduced infarction in the presence of cangrelor, a common clinically used P2Y12 inhibitor. (A) Area at risk (AAR) related to left ventricle area (LV); (B) Image of TTC staining; (C) Infarct size related to AAR; (D) LDH release; (E) Mean arterial pressure (MAP, mmHg) throughout the whole protocol; (F) glucose level at three time point. *P < 0.05 vs. Saline group, AAR/LV and IS/AAR data was analyzed by Student's t-test; LDH data was analyzed by Mann-Whitney U test; MAP and glucose level were analyzed using two-way Repeated Measures ANOVA applying the Bonferroni multiple comparisons test when relevant. Saline group (n = 7); NR group (n = 8).

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  Figure 6. NR lost protection against in vivo cardiac IR injury in presence of propofol. NR treatment failed to reduce infarction in the presence of propofol, a common clinically used anesthesia. (A) Area at risk (AAR) related to left ventricle area (LV); (B) Image of TTC staining; (C) Infarct size related to AAR; (D) LDH release; (E) Mean arterial pressure (mmHg) throughout the whole protocol; (F) glucose level at three time point. Data was analyzed by Student's t-test; *P < 0.05 vs. T1, ##P < 0.01 vs. T2, MAP and glucose level were analyzed using two-way Repeated Measures ANOVA applying the Bonferroni multiple comparisons test when relevant. Saline group (n = 6); NR group (n = 6).