Repetitive ischemia by coronary stenosis induces a novel window of ischemic preconditioning

Abstract

Background: The hypothesis of the present study was that molecular mechanisms differ markedly when mediating ischemic preconditioning induced by repetitive episodes of ischemia versus classic first- or second-window preconditioning.

Methods and results: To test this, chronically instrumented conscious pigs were subjected to either repetitive coronary stenosis (RCS) or a traditional protocol of second-window ischemic preconditioning (SWIPC). Lethal ischemia, induced by 60 minutes of coronary artery occlusion followed by reperfusion, resulted in an infarct size/area at risk of 6+/-3% after RCS and 16+/-3% after SWIPC (both groups P<0.05, less than shams 42+/-4%). Two molecular signatures of SWIPC, the increased expression of the inducible isoform of nitric oxide synthase and the translocation of protein kinase Cepsilon to the plasma membrane, were observed with SWIPC but not with RCS. Confirming this, pretreatment with a nitric oxide synthase inhibitor prevented the protection conferred by SWIPC but not by RCS. Microarray analysis revealed a qualitatively different genomic profile of cardioprotection between ischemic preconditioning induced by RCS and that induced by SWIPC. The number of genes significantly regulated was greater in RCS (5739) than in SWIPC (2394) animals. Of the 5739 genes regulated in RCS, only 31% were also regulated in SWIPC. Broad categories of genes induced by RCS but not SWIPC included those involved in autophagy, endoplasmic reticulum stress, and mitochondrial oxidative metabolism. The upregulation of these pathways was confirmed by Western blotting.

Conclusions: RCS induces cardioprotection against lethal myocardial ischemia that is at least as powerful as traditional ischemic preconditioning but is mediated through radically different mechanisms.

Figure 1

A, Changes in IZWT during IPC and recovery from IPC (ie, baseline before lethal CAO) in control (sham), SWIPC, RCS, SWIPC/L-NNA, and RCS/L-NNA pigs. Absolute values for IZWT are shown in parentheses, and the number of pigs in each group is noted. Note that SWIPC resulted in a complete loss of function but returned to baseline after IPC, whereas RCS only partially reduced IZWT, but function remained depressed after RCS was completed. B, Changes in IZWT during the lethal 60-minute period of CAO followed by 4 days of reperfusion in all 5 groups. Note that 60 minutes of CAO resulted in a similar reduction in IZWT in all groups, but recovery was different. The greatest recovery of IZWT occurred in the RCS groups, consistent with reduced infarct size data shown in Figure 2.

Figure 2

Reduction in infarct size conferred by SWIPC vs RCS. The Figure shows the reduction in IS/AAR in SWIPC and RCS compared with controls and shows that with addition of L-NNA, the cardioprotection was lost in SWIPC but not RCS. *P<0.05 vs control. n=5 per group except for SWIPC with L-NNA (n=7) and RCS with L-NNA (n=4). The dual perfusion of 1 pig heart in the RCS with L-NNA group was not adequate, and infarct size data could only be obtained in 4 animals in that group.

Figure 3

Mechanisms of SWIPC are not activated during RCS. Upregulation of iNOS, NOS activity, and the translocation of PKCε to the plasma membrane found during SWIPC are absent in the RCS model. *P<0.05 vs sham. Western blots show representative examples (n=5 for shams and n=6 for each SWIPC and RCS). C indicates cytosolic fraction; P, particulate fraction.

Figure 4

Differentially expressed genes from 2 models. A, Probe sets for genes that are significantly regulated (fold change >1.2 and q-value <0.05 by significance analysis of microarrays) in either SWIPC or RCS were selected, clustered, and shown in a heat map. The log2-based values were median-centered in each row and are represented according to the color scale shown at the bottom. B, Venn diagram that summarizes the similarities and differences between the 2 models using significant genes in Figure 4A. When multiple probe sets were present for a gene, the most significant one, either upregulated or downregulated, was selected. n=5 per group.

Figure 5

Significant GO entries were selected by the hypergeometric test (<0.05 after Bonferroni correction) for SWIPC and RCS models and are shown in a heat map with color representing the significance score (see Methods for its calculation). As shown in the color scale at the bottom, GO entries with positive significance scores (greater significant probability values for biased representation of upregulated genes) are shown in red, and those with negative scores (greater significant probability values for biased representation of downregulated genes) are shown in blue. To avoid redundancy in the graph, GO entries with >100 child entries are not shown. NF indicates nuclear factor; CoA, coenzyme A.

Figure 6

Regulation of proteins involved in cardioprotection specific to RCS. There was no significant change (data expressed as fold change from sham) in the expression of cathepsin D, E2F1, and GRP78 in the model of SWIPC compared with sham, but they were increased significantly by 20-, 4.5-, 4.0-, and 4.2-fold, respectively, in the RCS model (P>0.05 vs SWIPC). Cathepsin B was increased by 100-fold in RCS (P<0.01 vs SWIPC). Reciprocally, pyruvate dehydrogenase kinase and ATP synthase-β were downregulated in RCS but not in SWIPC. n=6 per group, except for cathepsins in RCS, n=7. *P<0.05 vs SWIPC.