Postconditioning: a form of "modified reperfusion" protects the myocardium by activating the phosphatidylinositol 3-kinase-akt pathway

Brief intermittent episodes of ischemia and reperfusion, at the onset of reperfusion after a prolonged period of ischemia, confer cardioprotection, a phenomenon termed "ischemic postconditioning" (Postcond). We hypothesized that this phenomenon may just represent a modified form of reperfusion that activates the reperfusion injury salvage kinase (RISK) pathway. Isolated perfused rat hearts were subjected to: (a) 35 minutes of ischemia and 120 minutes of reperfusion, and infarct size was determined by tetrazolium staining; or (b) 35 minutes of ischemia and 7 minutes of reperfusion, and the phosphorylation states of Akt, endothelial NO synthase (eNOS), and p70S6K were determined. Postcond reduced infarct size from 51.2±3.4% to 31.5±4.1% (P<0.01), an effect comparable with ischemic preconditioning (IPC; 27.5±2.3%; P<0.01). Of interest, the combined protective effects of IPC and Postcond were not additive (30.1±4.8% with IPC+Postcond; P=NS). Inhibiting phosphatidylinositol 3-kinase (PI3K) at reperfusion using LY or Wortmannin (Wort) during the first 15 minutes of reperfusion completely abolished Postcond-induced protection (31.5±4.1% with Postcond versus 51.7±4.5% with Postcond+LY, P<0.01; 56.2±10.1% with Postcond+ Wort; P<0.01), suggesting that Postcond protects the heart by activating PI3K-Akt. Western blot analysis demonstrated that Postcond induced a significant increase in phosphorylation of Akt, eNOS, and p70S6K in an LY- and Wort-sensitive manner. In conclusion, we show for the first time that ischemic Postcond protects the myocardium by activating the prosurvival kinases PI3K-Akt, eNOS, and p70S6K in accordance with the RISK pathway

Transient mitochondrial permeability transition pore opening mediates preconditioning-induced protection

Background - Transient (low-conductance) opening of the mitochondrial permeability transition pore (mPTP) may limit mitochondrial calcium load and mediate mitochondrial reactive oxygen species (ROS) signaling. We hypothesize that transient mPTP opening and ROS mediate the protection associated with myocardial preconditioning and mitochondrial uncoupling.Methods and Results - Isolated perfused rat hearts were subjected to 35 minutes of ischemia/ 120 minutes of reperfusion, and the infarct-risk-volume ratio was determined by tetrazolium staining. Inhibiting mPTP opening during the preconditioning phase with cyclosporine-A (CsA, 0.2 mumol/L) or sanglifehrin-A (SfA, 1.0 mumol/L) abolished the protection associated with ischemic preconditioning (IPC) ( 20.2 +/- 3.6% versus 45.9 +/- 2.5% with CsA, 49.0 +/- 7.1% with SfA; P < 0.001); and pharmacological preconditioning with diazoxide (Dzx, 30 mu mol/L) (22.1 +/- 2.7% versus 46.3 +/- 3.0% with CsA, 48.4 +/- 5.5% with SfA; P < 0.001), CCPA ( the adenosine A1-receptor agonist, 200 nmol/L) (24.9 +/- 4.5% versus 54.4 +/- 6.6% with CsA, 42.6 +/- 9.0% with SfA; P < 0.001), or 2,4-dinitrophenol (DNP, the mitochondrial uncoupler, 50 mu mol/L) (15.7 +/- 2.7% versus 40.8 +/- 5.5% with CsA, 34.3 +/- 3.1% with SfA; P < 0.001), suggesting that mPTP opening during the preconditioning phase is required to mediate protection in these settings. Inhibiting ROS during the preconditioning protocols with N-mercaptopropionylglycine (MPG, 1 mmol/L) also abolished the protection associated with IPC (20.2 +/- 3.6% versus 47.1 +/- 3.8% with MPG; P < 0.001), diazoxide (22.1 +/- 2.7% versus 56.3 +/- 3.8% with MPG; P < 0.001), and DNP (15.7 +/- 2.7% versus 50.7 +/- 6.6% with MPG; P < 0.001) but not CCPA (24.9 +/- 4.5% versus 26.5 +/- 8.4% with MPG; P = NS). Further experiments in adult rat myocytes demonstrated that diazoxide induced CsA-sensitive, low-conductance transient mPTP opening (represented by a 28 +/- 3% reduction in mitochondrial calcein fluorescence compared with control; P < 0.01).Conclusions - We report that the protection associated with IPC, diazoxide, and mitochondrial uncoupling requires transient mPTP opening and ROS

Myocardial protection by insulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell-survival signaling

The "metabolic cocktail" comprising glucose-insulin-potassium administrated at reperfusion reduces infarct size in the in vivo rat heart. We propose that insulin is the major component mediating this protection and acts via Akt prosurvival signaling. This hypothesis was studied in isolated perfused rat hearts (measuring infarct size to area of risk [%]) subjected to 35 minutes regional myocardial ischemia and 2 hours reperfusion. Insulin administered at the onset of reperfusion attenuated infarct size by 45% versus control hearts (P<0.001). Insulin-mediated cardioprotection was found to be independent of the presence of glucose at reperfusion. Moreover, the cell survival benefit of insulin is temporally dependent, in that insulin administration from the onset of reperfusion and maintained for either 15 minutes or for the duration of reperfusion reduced infarct size. In contrast, protection was abrogated if insulin administration was delayed until 15 minutes into reperfusion. Pharmacological inhibition of both upstream and downstream signals in the Akt prosurvival pathway abolished the cardioprotective effects of insulin. Here coadministration of insulin with the tyrosine kinase inhibitor lavendustin A, the phosphatidylinositol3-kinase (PI3-kinase) inhibitor wortmannin, and mTOR/p70s6 kinase inhibitor rapamycin abolished cardioprotection. Steady-state levels of activated/phosphorylated Akt correlated with insulin administration. Finally, downstream prosurvival targets of Akt including p70s6 kinase and BAD were modulated by insulin. In conclusion, insulin administration at reperfusion reduces myocardial infarction, is dependent on early administration during reperfusion, and is mediated via Akt and p70s6 kinase dependent signaling pathway. Moreover, BAD is maintained in its inert phosphorylated state in response to insulin therapy

Postconditioning protects against endothelial ischemia-reperfusion injury in the human forearm

Background: Hypoxic cell death follows interruption of blood supply to tissues. Although successful restoration of blood flow is mandatory for salvage of ischemic tissues, reperfusion can paradoxically place tissues at risk of further injury. Brief periods of ischemia applied at the onset of reperfusion have been shown to reduce ischemia-reperfusion (IR) injury, a phenomenon called postconditioning. The aim of this study was to determine whether postconditioning protects against endothelial IR injury in humans, in vivo. Methods and Results: Brachial artery endothelial function was assessed by vascular ultrasound to measure flow-mediated dilation (FMD) in response to forearm reactive hyperemia. FMD was measured before and after IR (20 minutes of arm ischemia followed by 20 minutes of reperfusion) in healthy volunteers. To test the protective effects of postconditioning, 3 cycles of reperfusion followed by ischemia (each lasting 10 or 30 seconds) were applied immediately after 20 minutes of arm ischemia. To determine whether postconditioning needs to be applied at the onset of reperfusion, a 1-minute period of arm reperfusion was allowed before the application of the 10-second postconditioning stimulus. IR caused endothelial dysfunction (FMD 9.1±1.2% pre-IR, 3.6±0.7% post-IR, P<0.001; n=11), which was prevented by postconditioning applied as 10-second cycles of reperfusion/ischemia (FMD 9.9±1.7% pre-IR, 8.3±1.4% post-IR, P=NS; n=11) and 30-second cycles of reperfusion/ischemia (FMD 10.8±1.7% pre-IR, 9.5±1.5% post-IR, P=NS; n=10) immediately at the onset of reperfusion. No protection was observed when the application of the 10-second postconditioning stimulus was delayed for 1 minute after the onset of reperfusion (FMD 9.8±1.2% pre-IR, 4.0±0.9% post-IR, P<0.001; n=8). Conclusions: This study demonstrates for the first time that postconditioning can protect against endothelial IR injury in humans. Postconditioning might reduce tissue injury when applied at the onset of reperfusion by modifying the reperfusion phase of IR

A new era in the management of type 2 diabetes: Is cardioprotection at long last a reality?

The EMPA-REG OUTCOME and the LEADER trials have revealed a new era in the management of type 2 diabetes. The SGLT2 inhibitor empagliflozin demonstrated a lower rate of the primary composite outcome of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke compared to placebo. Liraglutide, a GLP-1 analogue, succeeded to demonstrate reduction on a composite outcome including first occurrence of cardiovascular death, nonfatal myocardial infarction or non-fatal stroke. These two medications act through different mechanisms and has consequently shown different patterns of cardiovascular benefit. In one hand, empagliflozin showed an earlier effect compared to those observed using liraglutide. On the other hand, the difference between empagliflozin and placebo was driven by a significant reduction in death from cardiovascular causes, with and striking disconnect showing no significant between-group difference in the risk of myocardial infarction or stroke. In contrast, liraglutide reduced consistently all components of the composite endpoint. Based on the different temporal pattern of achieving clinical benefit one might flirt with the idea that liraglutide seems to provide a chronic “protection” that better fits in a longer metabolic effect with an impact in the progression of atherosclerosis, whilst empagliflozin provides an acute effect compatible with an immediate hemodynamic action. After years going from “bench to bedside” in order to discover the holy grail of cardioprotection, these 2 new studies suggest that we may have reached this state and it is time to go from “bed back to bench side” to understand the mechanisms of this potential paradigm shift

Exosomes and cardioprotection - A critical analysis

Exosomes are nano-sized vesicles released by numerous cell types that appear to have diverse beneficial effects on the injured heart. Studies using exosomes from stem cells or from the blood have indicated that they are able to protect the heart both in models of acute ischaemia and reperfusion, and during chronic ischaemia. In addition to decreasing initial infarct size, they are able to stimulate angiogenesis, reduce fibrosis and remodelling, alter immune cell function and improve long-term cardiac contractile function. However, since the technology and techniques used for the study of exosomes is relatively immature and continually evolving, there remain many important caveats to the interpretation of studies. This review presents a critical analysis of the field of exosomes and cardioprotection. We analyse the effects of exosomes from all types of stem cells investigated to date, summarize the major effects observed and their potential mechanism, and offer our perspective on the major outstanding issues

Glimepiride, a novel sulfonylurea, does not abolish myocardial protection afforded by either ischemic preconditioning or diazoxide

Background: The sulfonylurea glibenclamide (Glib) abolishes the cardioprotective effect of ischemic preconditioning (IP), presumably by inhibiting mitochondrial KATP channel opening in myocytes. Glimepiride (Glim) is a new sulfonylurea reported to affect nonpancreatic KATP channels less than does Glib. We examined the effects of Glim on IP and on the protection afforded by diazoxide (Diaz), an opener of mitochondrial KATP channels. Methods and results: Rat hearts were Langendorff-perfused, subjected to 35 minutes of regional ischemia and 120 minutes of reperfusion, and assigned to 1 of the following treatment groups: (1) control; (2) IP of 2x 5 minutes each of global ischemia before lethal ischemia; or pretreatment with (3) 30 µmol/L Diaz, (4) 10 µmol/L Glim, (5) 10 µmol/L Glib, (6) IP+Glim, (7) IP+Glib, (8) Diaz+Glim, or (9) Diaz+Glib. IP limited infarct size (18.5±1% vs 43.7±3% in control, P<0.01) as did Diaz (22.2±4.7%, P<0.01). The protective actions of IP or Diaz were not abolished by Glim (18.5±3% in IP+Glim, 22.3±3% in Diaz+Glim; P<0.01 vs control). However, Glib abolished the infarct-limiting effects of IP and Diaz. Patch-clamp studies in isolated rat ventricular myocytes confirmed that both Glim and Glib (each at 1 µmol/L) blocked sarcolemmal KATP currents. However, in isolated cardiac mitochondria, Glim (10 µmol/L) failed to block the effects of KATP opening by GTP, in contrast to the blockade caused by Glib. Conclusions: Although it blocks sarcolemmal currents in rat cardiac myocytes, Glim does not block the beneficial effects of mitochondrial KATP channel opening in the isolated rat heart. These data may have significant implications for the treatment of type 2 diabetes in patients with ongoing ischemic heart disease