James Mykytenko

Evidence that cardioprotection by postconditioning involves preservation of myocardial opioid content and selective opioid receptor activation

Opioids introduced at reperfusion (R) following ischemia (I) reduce infarct size much like postconditioning, suggesting the hypothesis that postconditioning increases cardiac opioids and activates local opioid receptors. Anesthetized male rats subjected to 30 min regional I and 3 h R were postconditioned with three cycles of 10 s R and 10 s reocclusion at onset of R. Naloxone (NL), its peripherally restricted analog naloxone methiodide, delta-opioid receptor (DOR) antagonist naltrindole (NTI), kappa-opioid receptor antagonist norbinaltorphimine (NorBNI), and mu-opioid receptor (MOR) antagonist H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) were administered intravenously 5 min before R. The area at risk (AAR) was comparable among groups, and postconditioning reduced infarct size from 57 +/- 2 to 42 +/- 2% (P < 0.05). None of the antagonists alone altered infarct size. All antagonists abrogated postconditioning protection at higher doses. However, blockade of infarct sparing by postconditioning was lost, since tested doses of NL, NTI, NorBNI, and CTAP were lowered. The efficacy of NorBNI declined first at 3.4 micromol/kg, followed sequentially by NTI (1.1), NL (0.37), and CTAP (0.09), suggesting likely MOR and perhaps DOR participation. Representative small, intermediate, and large enkephalins in the AAR were quantified (fmol/mg protein; mean +/- SE). I/R reduced proenkephalin (58 +/- 9 vs. 33 +/- 4; P < 0.05) and sum total of measured enkephalins, including proenkephalin, peptide B, methionine-enkephalin, and methionine-enkephalin-arginine-phenylalanine (139 +/- 17 vs. 104 +/- 7; P < 0.05) compared with shams. Postconditioning increased total enkephalins (89 +/- 8 vs. 135 +/- 5; P < 0.05) largely by increasing proenkephalin (33 +/- 4 vs. 96 +/- 7; P < 0.05). Thus the infarct-sparing effect of postconditioning appeared to involve endogenously activated MORs and possibly DORs, and preservation of enkephalin precursor synthesis in the AAR.

Inhibition of myocardial apoptosis by postconditioning is associated with attenuation of oxidative stress-mediated nuclear factor-κB translocation and TNFα release

ABSTRACT Oxidative stress-stimulated nuclear factor-κB (NF-κB) activation has been associated with rapid transcription of TNF-α and induction of apoptosis. This study tested the hypothesis that postconditioning (Postcon) reduces myocardial apoptosis and inhibits translocation of NF-κB and release of TNF-α secondary to an attenuation of oxidant generation during reperfusion. Anesthetized rats were subjected to 30 min of ischemia and 3 h of reperfusion and divided randomly to Control or Postcon (three cycles of 10-s reperfusion and 10-s reocclusion applied at the onset of reperfusion) group, respectively. Relative to Control, Postcon reduced the plasma malondialdehyde (1.21 ± 0.08 vs. 0.8 ± 0.06* μM/mL) and decreased the generation of superoxide radical in area at risk myocardium (dihydroethidium staining). Compared with Control, Postcon also inhibited translocation of NF-κB to nuclei (167% ± 21% vs. 142% ± 18%*), decreased the level of plasma TNF-α (1,994 ± 447 vs. 667 ± 130* pg/mL), and inhibited caspase-3 activity (0.57% ± 0.1% vs. 0.21% ± 0.1%*). The number of apoptotic cells (percent total nuclei) in ischemic myocardium was reduced (20% ± 1% vs. 11% ± 2%*), consistent with reduced appearance of DNA fragmentation. To support whether oxidant generation is important in the triggering of cytokine release and apoptosis, N-acetylcysteine (NAC), a potent antioxidant agent, was administered before ischemia and at reperfusion. Treatment with NAC inhibited superoxide radical generation and decreased plasma malondialdehyde to a comparable level to that in Postcon, concomitant with an inhibition of NF-κB expression (42% ± 8%*) and reduction of release of TNF-α (231 ± 72* pg/mL). Caspase-3 activity (0.33% ± 0.1%*) and apoptotic cells (12% ± 1%*) were also comparably reduced by NAC. These data suggest that Postcon attenuates myocardial apoptosis, reduces caspase-3 activity, and is potentially mediated by inhibiting oxidant-activated NF-κB-TNF-α signaling pathway. *P < 0.05 Postcon and NAC vs. Control.

Long-term inhibition of myocardial infarction by postconditioning during reperfusion.

AbstractCardioprotection with postconditioning has been well demonstrated after a short period of reperfusion. This study tested the hypothesis that postconditioning reduces infarct size, vascular dysfunction, and neutrophil accumulation after a long-term reperfusion. Canines undergoing 60 min left anterior descending artery (LAD) occlusion were divided into two control groups of either 3 h or 24 h of full reperfusion and two postconditioning groups with three 30 s cycles of reperfusion and re-occlusion applied at the onset of either 3 h or 24 h of reperfusion. Size of the area at risk (AAR) and collateral blood flow during ischemia were similar among groups. In controls, infarct size as percentage of the AAR (30 ± 3 vs. 39 ± 2* %) by TTC staining, superoxide anion generation from the post-ischemic coronary arteries by lucigenin-enhanced chemiluminescence [(89 ± 5 vs. 236 ± 27* relative light units (RLU/mg)], and neutrophil (PMN) accumulation by immunohistochemical staining in the AAR (52 ± 11 vs. 84 ± 14* cells/mm2 myocardium) significantly increased between 3 and 24 h of reperfusion. Postconditioning reduced infarct size (15 ± 4† and 27 ± 3.6† %), superoxide anion generation (24 ± 4† and 43 ± 11† RLU/mg), and PMN accumulation (19 ± 6† and 45 ± 8† cells/mm2 myocardium) in the 3 and 24 h reperfusion groups relative to time-matched controls. These data suggest that myocardial injury increases with duration of reperfusion; reduction in infarct size and attenuation in inflammatory responses with postconditioning persist after a prolonged reperfusion. * p < 0.05 24 vs. 3 h control; † p < 0.05 postconditioning vs. time-matched control.

Neutrophil inhibition contributes to cardioprotection by postconditioning

Postconditioning (postcon) reduces infarct size, myocardial superoxide (•O2) generation, and neutrophil (PMN) accumulation. It is unknown whether inhibition of PMNs influence cardioprotection by postcon. The present study tested the following hypotheses: (1) myocardial salvage by postcon is modified by inhibition of PMNs and (2) postcon directly inhibits PMN •O2 generation.