The major finding of the present investigation is that RCS induces cardioprotection that is quantitatively at least equal to that of the more traditionally used IPC protocols (SWIPC), but the mechanisms mediating this protection are radically different in the RCS model. Most importantly, it is widely accepted that PKC and NO are cornerstones of the cardioprotection induced by SWIPC2–7
and that pretreatment with an NOS inhibitor can abolish the IPC. These cardinal observations were confirmed in the model of SWIPC used in the present investigation. In contrast, the protection afforded by RCS was not accompanied by upregulation of either PKC or iNOS and was not diminished by pretreatment with the NOS inhibitor. Once we recognized that the mechanisms mediating IPC were so disparate to those induced by RCS, we conducted microarray analysis to determine the extent of differences in the genomic signature of SWIPC compared with RCS, with the hypothesis that novel mechanisms mediating RCS would be discovered with this approach.
Indeed, a possible explanation for these marked mechanistic differences may come from the comparison of the genomic profile between the models used in the present study. The present results indicate that the model of RCS regulates specific categories of genes that are not affected by SWIPC. This model-specific genomic regulation includes a downregulation of genes involved in mitochondrial energy metabolism and an upregulation of genes that participate in 3 pathways of cardiac cytoprotection, ie, autophagy, ER stress response, and cell survival ().
Multiple genes involved in mitochondrial oxidative function are downregulated by RCS, which is in agreement with a recent study conducted by 2D gel electrophoresis showing a similar downregulation of mitochondrial proteins in a model of long-term (3 months) coronary artery stenosis.17
Several studies have illustrated the importance of the production of reactive oxygen species from mitochondria to explain the mechanisms of IPC.18–20
It is therefore possible that a shutdown of mitochondrial function in repetitive ischemia will elicit the activation of reactive oxygen species–independent and totally different survival mechanisms. These alternative mechanisms include autophagy, ER stress response, and cell survival. At this point, we do not have definitive evidence of the extent of cardioprotection induced by these mechanisms. Future work will be required to elucidate the relative importance of these pathways, including mitochondrial mechanisms; however, this will be complicated by the possibility that several mechanisms mediating protection after RCS could be redundant. This would not be unexpected in view of the large number of genes (5739) that were found to be regulated in the heart in the RCS model.
Autophagy is an intracellular process of degradation of damaged organelles and denatured cytoplasmic proteins that reduces cellular stress and promotes cell survival.21
We recently showed that activation of the autophagy pathway of protein degradation is a survival mechanism in the chronically ischemic myocardium characterized by autophagosomes in myocytes, as seen with electron microscopy, and by upregulation of proteins involved in autophagy.9
Although autophagy can disrupt myocytes, the salvaged amino acids can be used to build new proteins, whereas necrosis and apoptosis result in cell death without regeneration. The lysosomal cathepsins B and D are central to the mechanisms of autophagy, because their deletion leads to an inhibition of autophagy and accumulation of denatured proteins.22
The present results confirm a major upregulation of both enzymes in the model of RCS, but this was not observed in SWIPC.
The ER stress response is a mechanism that improves the quality of protein translation by limiting the production and accelerating the degradation of denatured peptides by the proteasome.23
The ER is crucial for protein synthesis and secretion, but a high rate of translation automatically includes a large proportion of misfolded and denatured proteins that must be destroyed. ER-specific chaperones, such as GRP78, prevent the accumulation of unfolded proteins in the ER by promoting their translocation to the ubiquitin-proteasome system of protein degradation, a process known as ER-associated degradation.23
If the ER-associated degradation is saturated, there will be a rapid accumulation of unfolded proteins in the ER, which triggers the “unfolded protein response” that results in the death of the cell by apoptosis.23
In that respect, autophagy and ER stress response are complementary in preventing the accumulation of denatured proteins that would automatically increase cellular stress.
The mechanistic differences between SWIPC and RCS described in the present study have profound clinical implications. As discussed above, the model of RCS resembles more closely the clinical condition of patients with ventricular dysfunction that results from sequential episodes of stress-induced stable angina.24
Furthermore, although preconditioning has been the subject of intense investigation in various animal models, its relevance in the clinical setting is less established, which can be attributed in part to the fact that differences in mechanisms mediating cardioprotection are radically different in the presence of chronic, repetitive ischemia than after a traditional IPC stimulus applied to virgin myocardium.
One other difference between traditional IPC and the RCS-induced ischemic protection must be mentioned, ie, preconditioning generally but not always25–27
follows episodes of completed CAO and reperfusion and not coronary stenosis. Accordingly, we examined 3 additional pigs with a protocol of two 10-minute periods of CAO followed by CAR, which was repeated every 12 hours 6 times. IS/AAR was 12±3% (data not shown), similar to that observed with RCS (6±3%) or SWIPC (16±4%). After L-NNA, in 3 additional pigs with this protocol, ischemic protection was not abolished, ie, IS/AAR was 15±2%, similar to that observed with RCS and L-NNA (). Thus, although NOS is critical to the second window of IPC, it is not involved in mediating the IPC elicited by repetitive episodes of either coronary stenosis or complete coronary occlusion. However, this does not completely rule out a role for NO, because it was recently shown that NO can be produced in the ischemic heart independently from NOS activity.28
Another mechanism of IPC has been described with coronary microembolization over a 6-hour period.29
Microinfarction and inflammation occur, which results in IPC mediated by tumor necrosis factor-α. Even though this occurs temporally before SWIPC (6 hours versus 24 hours), it was termed a “third window” of IPC.25
The RCS model also is characterized by sparse, focal lesions of necrosis, primarily in the subendocardium.8
Therefore, it is possible that mechanisms related to inflammation may be involved in the cardioprotection, but as noted above, the microarray analysis identified 5739 genes regulated in the RCS model. Accordingly, it is not likely that 1 mechanism can be responsible for cardioprotection with chronic, repetitive episodes of ischemia.
In conclusion, RCS induces powerful protection against lethal myocardial ischemia, equivalent to that induced by traditional IPC but which acts through mechanisms radically different from those observed during preconditioning. These data demonstrate the existence of a novel “third window” of cardioprotection, which is likely involved in the protection inherent to chronic and repetitive ischemia found in patients with coronary artery disease.
Ischemic preconditioning (IPC), discovered 20 years ago, is the most powerful intervention known to protect myocardium, and yet it has proved difficult to translate the knowledge obtained into clinical therapy. The vast majority of experimental studies in IPC have used a brief episode of the IPC stimulus on a background of a normal heart with normal coronary arteries, ie, virgin ischemia. Our hypothesis is that the molecular mechanisms that mediate IPC are radically different in the setting of chronic ischemia, more akin to the situation in patients with coronary artery disease. To test this, conscious, chronically instrumented pigs were subjected to either repetitive coronary stenosis (RCS) or a traditional protocol of second-window IPC (SWIPC). Lethal ischemia, applied after IPC, resulted in similar reductions in infarct size/area at risk for animals in the RCS and SWIPC protocols. Two molecular signatures of SWIPC, the increased expression of the inducible isoform of NO synthase and the translocation of protein kinase Cε to the plasma membrane, were observed with SWIPC but not with RCS. Microarray analysis revealed a qualitatively different genomic profile of cardioprotection between IPC induced by RCS and that induced by SWIPC. The number of genes significantly regulated was greater in RCS than in SWIPC. Therefore, RCS induces cardioprotection against lethal myocardial ischemia that is at least as powerful as traditional IPC but is mediated through radically different mechanisms.