Mihaela M. Mocanu

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.

Protein kinase C. Its role in ischemic preconditioning in the rat.

The present study investigated whether protein kinase C (PKC) plays a role in ischemic preconditioning in the rat heart. Chelerythrine, a specific antagonist of PKC, and 1,2-dioctanoyl-sn-glycerol (DOG), a diacylglycerol analogue and specific antagonist of PKC, were used to determine whether preconditioning could be blocked or triggered, respectively. Sprague-Dawley rats were anesthetized and instrumented for coronary occlusion and reperfusion. All animals were subjected to 45 minutes of regional ischemia (ISC) followed by 2.5 hours of reperfusion. The preconditioning protocol consisted of 5 minutes of ischemia and then 10 minutes of reperfusion. There were six groups: (1) control (group C, n = 5), (2) preconditioned and ISC (group PC, n = 6), (3) chelerythrine given 2 minutes before ISC (group CC, n = 5), (4) preconditioned and chelerythrine given 2 minutes before ISC (group PCC, n = 6), (5) DOG (dissolved in dimethylsulfoxide [DMSO]) given 10 minutes before ISC (group CD, n = 5), and (6) DMSO given 10 minutes before ISC (group DMSO, n = 3). The end point was infarct size measured using triphenyl tetrazolium chloride and expressed as a percentage of the volume at risk (I/R), measured with fluorescent particles. I/R was significantly reduced by preconditioning (group C, 58.6 +/- 5.0%; group PC, 32.7 +/- 6.3%; P < .01) and by the PKC agonist DOG, which reduced I/R to a similar extent as preconditioning (group C, 58.6 +/- 5.0%; group CD, 28.0 +/- 7.0%; P < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Translating novel strategies for cardioprotection: the Hatter Workshop Recommendations

Ischemic heart disease (IHD) is the leading cause of death worldwide. Novel cardioprotective strategies are therefore required to improve clinical outcomes in patients with IHD. Although a large number of novel cardioprotective strategies have been discovered in the research laboratory, their translation to the clinical setting has been largely disappointing. The reason for this failure can be attributed to a number of factors including the inadequacy of the animal ischemia–reperfusion injury models used in the preclinical cardioprotection studies and the inappropriate design and execution of the clinical cardioprotection studies. This important issue was the main topic of discussion of the UCL-Hatter Cardiovascular Institute 6th International Cardioprotection Workshop, the outcome of which has been published in this article as the “Hatter Workshop Recommendations”. These have been proposed to provide guidance on the design and execution of both preclinical and clinical cardioprotection studies in order to facilitate the translation of future novel cardioprotective strategies for patient benefit.

Retrograde heart perfusion: The Langendorff technique of isolated heart perfusion

In the late 19th century, a number of investigators were working on perfecting isolated heart model, but it was Oscar Langendorff who, in 1895, pioneered the isolated perfused mammalian heart. Since that time, the Langendorff preparation has evolved and provided a wealth of data underpinning our understanding of the fundamental physiology of the heart: its contractile function, coronary blood flow regulation and cardiac metabolism. In more recent times, the procedure has been used to probe pathophysiology of ischaemia/reperfusion and disease states, and with the dawn of molecular biology and genetic manipulation, the Langendorff perfused heart has remained a stalwart tool in the study of the impact upon the physiology of the heart by pharmacological inhibitors and targeted deletion or up-regulation of genes and their impact upon intracellular signalling and adaption to clinically relevant stressful stimuli. We present here the basic structure of the Langendorff system and the fundamental experimental rules which warrant a viable heart preparation. In addition, we discuss the use of the isolated retrograde perfused heart in the model of ischaemia-reperfusion injury ex-vivo, and its applicability to other areas of study. The Langendorff perfusion apparatus is highly adaptable and this is reflected not only in the procedures longevity but also in the number of different applications to which it has been turned.

Caspase inhibition and limitation of myocardial infarct size: protection against lethal reperfusion injury.

Ischaemia‐reperfusion injury causes cell death by both necrosis and apoptosis. Caspase activation is a major event in apoptosis. We therefore examined the effect of caspase inhibitors during reperfusion upon myocardial infarction. Rat isolated hearts were subjected to 35 min coronary occlusion and 120 min reperfusion. Treatment groups were perfused with caspase inhibitors during early reperfusion. We assessed a non‐selective caspase inhibitor (Z‐VAD·fmk, 0.1 μM), a caspase‐8 inhibitor (Z‐IETD·fmk, 0.07 μM), a caspase‐9 inhibitor (Z‐LEHD·fmk, 0.07 μM) and a caspase‐3 inhibitor (Ac‐DEVD·cmk, 0.07 μM). All caspase inhibitors limited infarct size (infarct‐risk ratio per cent: control 38.5±2.6; Z‐VAD·fmk 24.6±3.4; Z‐LEHD·fmk 19.3±2.4; Z‐IETD·fmk 23.0±5.4; Ac‐DEVD·cmk 27.8±3.3; P<0.05 when compared with control value, 1‐way ANOVA). We conclude that caspase inhibition during early reperfusion protects myocardium against lethal reperfusion injury.

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 2× 5 minutes each of global ischemia before lethal ischemia; or pretreatment with (3) 30 &mgr;mol/L Diaz, (4) 10 &mgr;mol/L Glim, (5) 10 &mgr;mol/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 &mgr;mol/L) blocked sarcolemmal KATP currents. However, in isolated cardiac mitochondria, Glim (10 &mgr;mol/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.

Leptin, the obesity‐associated hormone, exhibits direct cardioprotective effects

Background and purpose: Protection against ischaemia‐reperfusion (I/R) injury involves PI3K‐Akt and p44/42 MAPK activation. Leptin which regulates appetite and energy balance also promotes myocyte proliferation via PI3K‐Akt and p44/42 MAPK activation. We, therefore, hypothesized that leptin may also exhibit cardioprotective activity.

Glucagon like peptide-1 is protective against myocardial ischemia/reperfusion injury when given either as a preconditioning mimetic or at reperfusion in an isolated rat heart model

A number of recent studies have identified potential beneficial effects for Glucagon Like Peptide-1 (GLP-1), a potent gut incretin hormone with notable cell regulatory and anti-apoptotic actions predominantly in the pancreas [1]. However, recent studies have not only identified receptors for GLP-1 in the myocardium [2] but also demonstrated its beneficial roles in cardiac physiology and metabolism. Investigators reported improved left ventricular performance and reduced stunning in both clinical and experimental studies [3]. In animal models, it was demonstrated that an activation of glycolysis [4] as well as a decrease in pyruvate and lactate [5] occur in the myocardium when GLP-1 is given prior to ischemia. We have recently shown GLP-1 protects the myocardium by reducing infarct size [6] in both an in vitro and in vivo rat heart model, when the agent was given throughout ischemia and reperfusion. This protective effect was mediated by GLP-1 receptor activation in addition to activation of the prosurvival kinases PI3K/Akt, p44/42 and PKA [6]. Since it is known that activation of the above kinases form part of the so-called Reperfusion Injury Salvage Kinase pathway (RISK pathway) [7], which is also associated with both preconditioning protection [8] as well as protection against reperfusion injury [7] we hypothesised that since GLP-1 has the ability to activate these kinases, it may protect against infarction when given either before ischemia (as a preconditioning mimetic) or at reperfusion.

Heat shock protein 27 protects the heart against myocardial infarction

Abstracts.Heat shock proteins (hsp) represent a group of chaperones which protects the cells against a diversity of stresses. It has been demonstrated that hsp27 is constitutively present in cells where it plays an important role in different cytoprotective processes which ultimately inhibit cell death. We investigated the response of the isolated perfused mouse heart over expressing hsp27 to the ischaemia/reperfusion injury using infarct size as an end point. Our results show for the first time that mice over expressing hsp27 (verified by Western blotting analysis) were found to be protected from lethal ischaemia/reperfusion injury compared to their negative littermates.

Enhancing AMPK activation during ischemia protects the diabetic heart against reperfusion injury

AMPK activation during ischemia helps the myocardium to cope with the deficit of energy production. As AMPK activity is considered to be impaired in diabetes, we hypothesized that enhancing AMPK activation during ischemia above physiological levels would protect the ischemic diabetic heart through AMPK activation and subsequent inhibition of mitochondrial permeability transition pore (mPTP) opening. Isolated perfused hearts from normoglycemic Wistar or diabetic Goto-Kakizaki (GK) rats (n ≥ 6/group) were subjected to 35 min of ischemia in the presence of 10, 20, and 40 μM of A-769662, a known activator of AMPK, followed by 120 min of reperfusion with normal buffer. Myocardial infarction and AMPK phosphorylation were assessed. The effect of A-769662 on mPTP opening in adult cardiomyocytes isolated from both strains was also determined. A-769662 at 20 μM reduced infarct size in both Wistar (30.5 ± 2.7 vs. 51.8 ± 3.9% vehicle; P < 0.001) and GK hearts (22.7 ± 3.0 vs. 48.5 ± 4.7% vehicle; P < 0.001). This protection was accompanied by a significant increase in AMPK and GSK-3β phosphorylation. In addition, A-769662 significantly inhibited mPTP opening in both Wistar and GK cardiomyocytes subjected to oxidative stress. We demonstrate that AMPK activation during ischemia via A-769662 reduces myocardial infarct size in both the nondiabetic and diabetic rat heart. Furthermore, this cardioprotective effect appears to be mediated through inhibition of mPTP opening. Our findings suggest that improving AMPK activation during ischemia can be another mechanism for protecting the ischemic heart.