Considerable attention has focused on the role of protein kinase C (PKC) in triggering the profound infarct-sparing effect of ischemic preconditioning (PC). In contrast, the involvement of inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)], the second messenger generated in parallel with the diacylglycerol-PKC pathway, remains poorly understood. We hypothesized that, if Ins(1,4,5)P(3) signaling [i.e., release of Ins(1,4,5)P(3) and subsequent binding to Ins(1,4,5)P(3) receptors] contributes to PC-induced cardioprotection, then the reduction of infarct size achieved with PC would be attenuated in mice that are deficient in Ins(1,4,5)P(3) receptor protein. To test this concept, hearts were harvested from 1) B6C3Fe-a/a-Itpr-1(opt+/-)/J mutants displaying reduced expression of Ins(1,4,5)P(3) receptor-1 protein, 2) Itpr-1(opt+/+) wild types from the colony, and 3) C57BL/6J mice. All hearts were buffer-perfused and randomized to receive two 5-min episodes of PC ischemia, pretreatment with d-myo-Ins(1,4,5)P(3) [sodium salt of native Ins(1,4,5)P(3)], the mitochondrial ATP-sensitive K(+) channel opener diazoxide, or no intervention (controls). After the treatment phase, all hearts underwent 30-min global ischemia followed by 2 h of reperfusion, and infarct size was delineated by tetrazolium staining. In both wild-type and C57BL/6J cohorts, area of necrosis in hearts that received PC, d-myo-Ins(1,4,5)P(3), and diazoxide averaged 28-35% of the total left ventricle (LV), significantly smaller than the values of 52-53% seen in controls (P < 0.05). In contrast, in Itpr-1(opt+/-) mutants, protection was only seen with diazoxide: neither PC nor d-myo-Ins(1,4,5)P(3) limited infarct size (52-58% vs. 56% of the LV in mutant controls). These data provide novel evidence that Ins(1,4,5)P(3) signaling contributes to infarct size reduction with PC.

Prophylactic treatment with D-myo-inositol 1,4,5-trisphosphate hexasodium [D-myo-Ins(1,4,5)P3], the sodium salt of the endogenous second messenger Ins(1,4,5)P3, triggers a reduction of infarct size comparable in magnitude to that seen with ischemic preconditioning (PC). However, the mechanisms underlying D-myo-Ins(1,4,5)P3-induced protection are unknown. Accordingly, our aim was to investigate the role of four archetypal mediators implicated in PC and other cardioprotective strategies (i.e., PKC, PI3-kinase/Akt, and mitochondrial and/or sarcolemmal K(ATP) channels) in the infarct-sparing effect of D-myo-Ins(1,4,5)P3. Fifteen groups of isolated buffer-perfused rabbit hearts [5 treated with D-myo-Ins(1,4,5)P3, 5 treated with PC, and 5 control cohorts] underwent 30 min of coronary artery occlusion and 2 h of reflow. One set of control, D-myo-Ins(1,4,5)P3, and PC groups received no additional treatment, whereas the remaining sets were infused with chelerythrine, LY-294002, 5-hydroxydecanoate (5-HD), or HMR-1098 [inhibitors of PKC, PI3-kinase, and mitochondrial and sarcolemmal ATP-sensitive K+ (K(ATP)) channels, respectively]. Infarct size (delineated by tetrazolium staining) was, as expected, significantly reduced in both D-myo-Ins(1,4,5)P3- and PC-treated hearts versus controls. D-myo-Ins(1,4,5)P3-induced cardioprotection was blocked by 5-HD but not HMR-1098, thereby implicating the involvement of mitochondrial, but not sarcolemmal, K(ATP) channels. Moreover, the benefits of D-myo-Ins(1,4,5)P3 were abrogated by LY-294002, whereas, in contrast, chelerythrine had no effect. These latter pharmacological data were corroborated by immunoblotting: D-myo-Ins(1,4,5)P3 evoked a significant increase in expression of phospho-Akt but had no effect on the activation/translocation of the cardioprotective epsilon-isoform of PKC. Thus PI3-kinase/Akt signaling and mitochondrial K(ATP) channels participate in the reduction of infarct size afforded by prophylactic administration of D-myo-Ins(1,4,5)P3.

Emerging evidence suggests that restoration of blood flow in a stuttering manner may limit lethal myocardial ischemia-reperfusion injury. However, the mechanisms contributing to this phenomenon, termed postconditioning (post-C), remain poorly defined. Our aim was to test the hypothesis that activation of classic "survival kinases," phosphatidylinositol 3-kinase (PI3-kinase) and/or extracellular signal-regulated kinase (ERK)1/2, may play a role in post-C-induced cardioprotection. In protocol 1, isolated buffer-perfused rabbit hearts underwent 30 min of sustained coronary artery occlusion and were randomized to receive abrupt reperfusion (controls) or four cycles of 30 s of reperfusion and 30 s of reocclusion before full restoration of flow (post-C). Protocol 2 was identical except control and postconditioned hearts received the PI3-kinase inhibitor LY-294002 (protocol 2A) or the ERK1/2 antagonist PD-98059 (protocol 2B) throughout the first 25 min of reperfusion, whereas in protocol 3, myocardial samples were obtained during the early minutes of reflow from additional control, postconditioned, and nonischemic sham hearts for the assessment, by standard immunoblotting, of phospho-Akt (downstream target of PI3-kinase) and phospho-ERK. Protocols 1 and 2 corroborated that infarct size (delineated by tetrazolium staining and expressed as a percent of risk region) was reduced in postconditioned hearts vs. control hearts and also revealed that post-C-induced cardioprotection was maintained despite LY-294002 treatment but was abrogated by PD-98059. These pharmacological data were supported by protocol 3, which showed increased immunoreactivity of phospho-ERK but not phospho-Akt with post-C. Thus our results implicate the involvement of ERK1/2 rather than PI3-kinase/Akt in the reduction of infarct size achieved with post-C.

Pretreatment with D-myo-inositol-1,4,5-trisphosphate hexasodium (D-myo-IP(3)), the sodium salt of the second messenger inositol 1,4,5-trisphosphate (IP(3)), is cardioprotective and triggers a reduction of infarct size comparable in magnitude to that obtained with ischemic preconditioning. However, this observation is enigmatic; whereas IP(3) signaling is conventionally initiated by receptor binding, IP(3) receptors are typically considered to be intracellular, and D-myo-IP(3) is membrane-impermeable. We propose that this paradox is explained by the presence of poorly characterized external IP(3) receptors and hypothesize that: 1) infarct size reduction with D-myo-IP(3) is receptor-mediated; and 2) communication via gap junctions and/or hemichannels is required to initiate this protection. To investigate the role of receptor binding, isolated buffer-perfused rabbit hearts underwent 30 min of coronary occlusion (CO) and 2 h of reflow. Prior to CO, hearts received no treatment (controls), D-myo-IP(3), L-myo-IP(3) (enantiomer not recognized by the IP(3) receptor), D-myo-IP(3) + the IP(3) receptor inhibitor xestospongin C (XeC), or XeC alone. Infarct size, assessed by tetrazolium staining, was reduced with D-myo-IP(3) treatment, whereas hearts that received L-myo-IP(3) or D-myo-IP(3) + XeC showed no protection. To evaluate the contribution of gap junctions/hemichannels, additional control and D-myo-IP(3)-treated cohorts received a 5-min infusion of heptanol or Gap 27, two structurally distinct gap junction inhibitors, administered at doses confirmed to attenuate intercellular transmission of a gap junction-permeable fluorescent dye. There was no infarct-sparing effect of D-myo-IP(3) in inhibitor-treated hearts. These data support the concepts that infarct size reduction with D-myo-IP(3) is triggered by receptor binding and that communication via gap junctions/hemichannels is involved in initiating this protection.