Repeated Remote Ischemic Postconditioning Protects Against Adverse Left Ventricular Remodeling and Improves Survival in a Rat Model of Myocardial InfarctionNovelty and Significance
Rationale: Remote ischemic conditioning induced by repeated episodes of transient limb ischemia is a clinically applicable method for protecting the heart against injury at the time of reperfusion.
Objective: To assess the effect of chronic, repeated, remote conditioning on infarct size and long-term remodeling after myocardial infarction.
Methods and Results: Rats with ischemia/reperfusion injury received different protocols of remote limb conditioning. While a single early episode of remote ischemic conditioning during coronary occlusion (perconditioning) resulted in a decrease in infarct size on both day 4 and day 28, when it was repeated (postconditioning) intermittently (every 3 days) and intensively (every day), it was not associated with a further decrease in infarct size. However, the protection against adverse remodeling offered by a single episode of limb perconditioning was further enhanced by repeated remote postconditioning therapy in a dose-dependent manner. In separate experiments there was a dose-dependent improvement in survival at 84 days by Kaplan–Meier analysis.
Conclusions: Whereas a single early episode of remote perconditioning reduces infarct size, repeated remote postconditioning further reduces adverse LV remodeling and improves survival in a dose-dependent fashion. These data may have clinical implications for the treatment of patients with evolving myocardial infarction.
Despite the timely application of reperfusion therapy, survivors of acute myocardial infarction (MI) are at significant risk from later myocardial remodeling, leading to the development of heart failure and elevated risk of death during 5-year follow-up.1,2 It is generally agreed that the acute inflammatory process that occurs early after acute MI (a prerequisite for adequate healing and scar formation3) can be adverse if persistent in the later post-MI recovery period. Continued oxidative stress results in proinflammatory cytokine release, cardiomyocyte apoptosis, ventricular fibrosis, and hypertrophy.4
Remote ischemic conditioning reduces infarct size when applied both before (preconditioning) and during (perconditioning) experimental and clinical MI.5,6 Similarly, local postconditioning can reduce infarct size.7 Clinically, postconditioning is achieved by repeated inflation and deflation of the angioplasty balloon after emergency coronary intervention.8 It is now known that postconditioning can be induced experimentally and in humans, remotely, using a transient limb ischemia stimulus identical to that of remote pre- and preconditioning, and in a recent study, remote postconditioning was more effective than local postconditioning in experimental MI9. We have shown that repeated daily remote conditioning by limb ischemia leads to significant downregulation of neutrophil activation and proinflammatory responses in humans.10 Thus, in the present study we investigated (1) whether a single episode of remote perconditioning protects against late remodeling; (2) whether a further decrease in infarct size or improved remodeling can be achieved when chronic remote postconditioning is given in addition to early preconditioning; and (3) how pathological processes involved in remodeling—including oxidative stress, inflammatory responses, and fibrotic and hypertrophic signaling—are modulated by repeated remote postconditioning.
Details of the study groups, experiment protocol, infarct size quantification, hemodynamic evaluation, and analytic methods are provided in the Online Data Supplement at http://circres.ahajournals.org. In brief, we established myocardial infarction by 45 minutes of LAD ligation, followed by reperfusion (MI group). Additional groups of animals (Figure 1A) were subjected to a single episode of remote perconditioning during ischemia (4 cycles of 5 minutes of hindlimb ischemia, 5 minutes of reperfusion, PerC group), perconditioning plus postconditioning (4 cycles of 5 minutes of hindlimb ischemia, 5 minutes of reperfusion) every 3 days for 28 days (D-3PostC group), and perconditioning plus postconditioning every day for 28 days (D-1PostC group). Different groups of animals were euthanized at 7 and 28 days and comparison was made with a sham-operated group, and a group subjected to daily sodium pentobarbital anesthesia (to control for daily intervention, SP group). In separate experiments, Kaplan–Meier survival was assessed in the same intervention groups (50 animals per group).
Details are provided in the Online Data Supplement at http://circres.ahajournals.org.
PerC, D-3PostC, and D-1PostC resulted in improved survival rate (Figure 1B) in comparison with MI and SP groups (P<0.05, for all). Interestingly, the improved survival was apparent as early as 28 days after MI only in the D-1PostC group, whereas in the D-3PostC group this effect was not observed until 56 days after MI. Furthermore, on day 84, D-1PostC was associated with improved survival in comparison with PerC and D-3PostC treatments (P<0.05, respectively).
Reduced Infarct Size
Infarct size was quantified for the 6 groups at 72 hours and at 28 days, with a similar pattern of response at each time point (Figure 2A through 2D). There was a reduction in infarct size, resulting in all conditioning groups in comparison with MI (P<0.05, respectively), but no difference between the conditioning groups (P>0.05, respectively).
There was intensive infiltration by both macrophages and neutrophils detected in the MI group, which was attenuated in PerC rats (P<0.01 versus MI group), and further attenuated by D-3PostC and D-1PostC (P<0.01 versus PerC group, respectively) with the greatest effect detected in D-1PostC group rats (P<0.05 versus D-3PostC group). The expression level of MCP-1 in the infarction border zone on day 4 showed the same pattern, demonstrating a dose-dependent effect in the remote conditioning groups (P<0.05, respectively) (Figure 3E).
Oxidative Stress, NF-κB Activation, and Cytokine Expression
The concentration of MDA, and phosphorylation of the NF-κB subunit p65 and its inhibitory protein IκBα, was significantly increased in MI rats on day 28, and was significantly reduced by per- and postconditioning in a dose-dependent fashion (Online Figure IA through IC). Interestingly, on day 28, PerC did not modify TNF-α and IL-1β levels (P>0.05, respectively), which were only attenuated by both D-3PostC and D-1PostC groups (P<0.01 vs MI group, respectively), however, to the same degree (P>0.05) (Online Figure ID and IE).
The reduction of TGF-β1/Smad2 signaling activation in the infarct borderzone was consistent with the beneficial pattern of NF-κB signaling activation (Online Figure IIA and IIB), and further supported by attenuated interstitial fibrosis (Online Figure IIC through IIE). When TGF-β1/Smad2 signaling activation was quantified in remote heart tissue, D-1PostC also showed the greatest effect (Online Figure IIIA and IIIB).
There was increased LV mass in the MI group, which was attenuated by PerC (P<0.05), with a further decrease with both D-3PostC and D-1PostC (P<0.05 versus PerC group, respectively), with the lowest mass observed in D-1PostC (P<0.001, versus D-3PostC group). Hypertrophy-related gene expression of ANP and β-MHC was attenuated, whereas the decrease in expression of α-MHC was recovered by remote conditioning, again in a dosage-dependent fashion (Online Figure IVA through IVC).
Improved Cardiac Geometry, Function, and Hemodynamics
On day 28, MI rats demonstrated significant LV dilation and decreased fractional shortening in comparison with sham rats (both P<0.05). Although PerC significantly improved LV remodeling in comparison with MI rats (P<0.05, respectively), repeated remote PostC therapy resulted in a further improvement in LV chamber size and function, with the greatest effects achieved by D-1PostC in comparison with D-3PostC (P<0.05, respectively) (Figure 4,A through 4D). Hemodynamic analysis demonstrated the same dose-dependent pattern of improvement (Online Figure VA through VC).
This is the first study to demonstrate that remote ischemic conditioning not only reduces infarct size but improves late LV remodeling and survival after MI. Our data show that a single episode of remote perconditioning affords some long-term protection, but repeated remote postconditioning during the 28 days post-MI significantly improves adverse LV remodeling and function and survival in dose-dependent fashion, and was closely associated with attenuated myocardial inflammatory responses and oxidative stress.
Role of Chronic Remote Postconditioning
Since the phenomenon of ischemic preconditioning was first described by Murry et al,11 many studies have been performed to elucidate the mechanisms by which pre- and postconditioning induce cardioprotection. Although the exact mechanisms are not fully understood, it is generally agreed that the attenuation of reactive oxygen species (ROS) generation12,–,14 is of paramount importance in the protection afforded by these strategies. Furthermore, it is well established that continued ROS generation and inflammation is pivotal in the process of post-MI remodeling.15,16 Remote conditioning has additional effects on circulating monocytes, downregulating white cell proinflammatory pathways,17 and when delivered daily for 10 days, reducing neutrophil adhesion, phagocytosis, and proinflammatory cytokine responses.10 Our findings are consistent with reduced myocardial oxidative stress (and hence reduced NF-kB phosphorylation), decreased inflammatory cell migration into the infarct zone (observed directly as attenuated MPO and ED-1 immunostaining density), and reduced local inflammatory cytokine signaling (reduced tissue MCP-1 expression, reduced circulating TNF-α and IL-1β levels). Local chemokines such as MCP-1 are responsible for inducing recruitment of mononuclear cells. Moreover, activated NF-κB pathways can also up-regulate the target gene expression of TNF-α and IL-1β.18 Although this modification of the local and circulating inflammatory milieu is likely crucial to the beneficial effects of chronic remote conditioning, more studies are required to dissect the mechanisms and assess whether it might be an adjunct to, or replace, current antiremodeling drug regimes frequently used after MI. Nonetheless, these findings have obvious relevance to post-MI recovery in humans. In this regard, and similar to other remote conditioning protocols, there is capacity for our observations to translate rapidly to clinical trials.19
Limitations of the Study
We observed that, on day 28, PerC rats did not show a decrease in TNF-α and IL-1β expression, whereas both D-3PostC and D-1PostC showed a significant decrease in chemokine levels. This could reflect a dosage-dependent response; however, we also observed a decrease in NF-κB phosphorylation in the PerC group. Consequently, it is difficult to explain the apparent conflicting data on the basis of our data.
Although the data of improved survival rate were clear, we did not include any assessment of remodeling after cessation of repeated PostC. It is possible that the time course of adverse remodeling was simply delayed, rather than permanently modified.
We have shown that chronic remote ischemic postconditioning provides dose-dependent protection against pathological ventricular remodeling, and improves survival after myocardial infarction.
Sources of Funding
The study was supported by the Canadian Institutes for Health Research and the Leducq Foundation (to A.N.R.), by institutional financial support of Shanghai Jiao Tong University Sixth Hospital (to W.Z.), and partly by the Natural Science Foundation of China (No. 81070110; to M.W.).
In February 2011, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.7 days.
Non-standard Abbreviations and Acronyms
- daily remote ischemic postconditioning for 28 days
- remote ischemic postconditioning every 3 days for 28 days
- remote ischemic perconditioning
- Received November 4, 2010.
- Revision received March 25, 2011.
- Accepted March 28, 2011.
- © 2011 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Remote ischemic preconditioning induced by transient limb ischemia reduces myocardial infarct (MI) size in animals and humans.
Remote ischemic preconditioning reduces proinflammatory neutrophil gene expression, and when repeated, daily postconditioning reduces neutrophil adhesion and phagocytosis.
What New Information Does This Article Contribute?
Remote postconditioning administered after MI does not lead to further reduction in infarct size.
Remote postconditioning administered after myocardial infarction in rats reduces adverse left ventricular (LV) remodeling at 28 days in a dose-dependent fashion.
Improved remodeling is associated with reduced peri-infarct inflammation and fibrosis.
Remote postconditioning administered for 28 days after MI in rats improves survival at 12 weeks in a dose-dependent fashion
While the beneficial early effects of remote ischemic conditioning (induced by 4 cycles of 5 minutes of transient limb ischemia followed by reperfusion) on MI are well established, its effect when administered repeatedly after MI (remote postconditioning) has not been examined. We show here, for the first time, that remote postconditioning administered every 3 days, or every day, for 28 days improves post-MI LV remodeling at 28 days, and survival at 12 weeks, in a dose-dependent fashion. This simple technique is readily applicable to humans after MI and may lead to significant benefits in post-MI morbidity and mortality.