Impact of age and sex on response to ischemic preconditioning in the rat heart: differential role of the PI3K-AKT pathway.

Sex and aging represent important factors that determine morbidity and mortality due to cardiovascular diseases in the human population. This study aimed to investigate the impact of aging on the response to ischemia-reperfusion in male and female rat hearts, and to explore a potential role of the PI3K-Akt pathway in the cardioprotective effects of ischemic preconditioning (IPC) in the myocardium of younger and older adult males and females. Langendorff-perfused nonpreconditioned and preconditioned hearts of 12- and 18-week-old male and female Wistar rats were subjected to regional ischemia and reperfusion with or without prior perfusion with the PI3K inhibitor wortmannin for the evaluation of ischemia-induced arrhythmias and the size of myocardial infarction (infarct size; IS). Aging did not modify IS in both sexes; however, it markedly increased susceptibility to arrhythmias. Although IPC effectively reduced IS in males and females of both ages, only the hearts of males and 18-week-old females benefited from its antiarrhythmic effect. In the preconditioned 12-week-old females, but not the 18-week-old females, and in males of both ages, wortmannin blunted the anti-infarct effect of IPC. In conclusion, activation of the PI3K-Akt pathway plays an important role in protection against lethal injury conferred by IPC in males irrespective of age. The IS-limiting effect of IPC appears to be PI3K-Akt-dependent only in the 12-week-old females.

The role of PPAR in myocardial response to ischemia in normal and diseased heart.

Peroxisome proliferator-activated receptors (PPAR), ligand-activated transcription factors, belong to the nuclear hormone receptor superfamily regulating expression of genes involved in different aspects of lipid metabolism, inflammation and cardiac energy production. Activation of PPAR-α isoform by its natural ligands, fatty acids (FA) and eicosanoids, promotes mitochondrial FA oxidation as the primary ATP-generating pathway. On the other hand, PPAR-γ regulates lipid anabolism or storage, while, until recently, the function of PPAR-ß/δ has been less explored. Under conditions associated with acute or chronic oxygen deprivation, PPAR-α modulates expression of genes that determine substrate switch (FA vs. glucose) aimed at maintenance of basic cardiac function. Although PPAR-α and PPAR-γ synthetic agonists, hypolipidemic and antidiabetic drugs, have been reported to protect the heart against ischemia/reperfusion injury, it is still a matter of debate whether PPAR activation plays a beneficial or detrimental role in myocardial response to ischemia, in particular, in pathological conditions. This article reviews some findings demonstrating the impact of PPAR activation on cardiac resistance to ischemia in normal and pathologically altered heart. Specifically, it addresses the issue of susceptibility to ischemia in the diabetic myocardium, with particular regards to the role of PPAR. Finally, involvement of PPAR in the mechanisms of lipid-independent cardioprotective effects of some hypolipidemic drugs is also discussed.

Activation of Akt kinase accompanies increased cardiac resistance to ischemia/reperfusion in rats after short-term feeding with lard-based high-fat diet and increased sucrose intake.

High-fat or high-carbohydrate food consumption contributes to changes in myocardial tolerance to ischemia. However, with respect to experimental models, most studies used diets with very high doses of cholesterol, saturated fatty acids, or fructose. In our study, we fed rats a high-fat diet based on lard in combination with administration of a sweet beverage (30% sucrose solution) (high-fat sucrose diet [HFS]). This diet was used to simulate the unhealthy dietary habit typical for developed countries. We hypothesized that the application of HFS diet for 48 days might initiate progression of pathologic changes in the heart associated with myocardial remodeling and activation of adaptive mechanisms. We investigated the influence of HFS diet on cardiac function and vulnerability to ischemia-reperfusion (I/R) injury in Langendorff-perfused rat hearts subjected to 30-minute global ischemia and 120-minute reperfusion as well as on Akt kinase and matrix metalloproteinases. We found lower food consumption in HFS group compared with controls, but a significant increase in visceral fat mass and concentrations of triacylglycerol, low-density lipoprotein, and very low-density lipoprotein cholesterol. Baseline heart functional parameters and their postischemic recovery were not affected by HFS diet. On the other hand, hearts of HFS group were more resistant to lethal I/R injury manifested by significantly smaller infarct size. In addition, there was lower content of collagen I and III in the left ventricle associated with Akt kinase activation and matrix metalloproteinase 9 up-regulation. In conclusion, feeding rats with HFS diet resulted in heart remodeling associated with activation of some adaptive mechanisms, which can contribute to modulation of myocardial resistance to I/R injury.

Influence of ischemia/reperfusion and modulation of PI3K/Akt kinase pathway on matrix metalloproteinase-2 in rat hearts.

Matrix metalloproteinases (MMPs) are enzymes that play an important role in degradation and remodeling of extracellular matrix and MMP-2 has been also shown as a primary mediator of the acute mechanical dysfunction of the heart immediately after ischemia/reperfusion (I/R). The aims of the study were to investigate the influence of I/R on MMP-2 and to study the effects of wortmannin on modulation of MMP-2 activities after cycle of short I/R procedures (ischemic preconditioning, IP). Wortmannin is a specific inhibitor of PI3K/Akt kinase pathway activation of which was found to play a role in infarct size limiting mechanisms in the rat heart. In the study isolated Langendorff-perfused rat hearts subjected to protocols of prolonged (test) I/R and/or IP were used. Wortmannin was infused before and during the reperfusion phase of IP. The levels and activation of proteins were determined by immunoblot assay. The MMP-2 activities were measured by zymography. We found that ischemia induced time-dependent activation of tissue pro-MMP-2. Strong activation occurred after 15 min ischemia, during prolonged ischemia and following reperfusion the activities of this form of MMP-2 declined. The specific activities of both 72 and 63 kDa forms of MMP-2 were increased in perfusates collected during reperfusion after 30 min ischemia and these activities peaked in the first minute of reperfusion. Cycle of short ischemia and reperfusion that led to increased cardiac tolerance against prolonged I/R reduced 72 kDa MMP-2 activities and induced also an activation of Akt kinase. The application of wortmannin was connected with inhibition of IP-mediated Akt kinase activation. Moreover, the actions of wortmannin were linked with modulation of MMP-2 activities. Our results suggest that MMP-2 may be involved in the responses of rat hearts to ischemia and point to possible relationship between Akt kinase and modulation of MMP-2 activities in rat hearts.

Mitochondrial KATP opening confers protection against lethal myocardial injury and ischaemia-induced arrhythmias in the rat heart via PI3K/Akt-dependent and -independent mechanisms.

Opening of mitochondrial KATP channels (mitoKATP) has been reported to underlie protection against ischaemia-reperfusion injury induced by ischaemic preconditioning (I-PC); however, the molecular mechanisms of its antiarrhythmic effect have not been fully elucidated. We explored the involvement of phosphatidylinositol 3-kinase (PI3K)/Akt in the PC-like effect of mitoKATP opener diazoxide with particular regard to its role in protection against ischaemia-induced arrhythmias. Langendorff-perfused rat hearts were subjected to 30 min LAD occlusion with or without a prior 15 min of perfusion with diazoxide (50 micromol/L) given either alone (D-PC) or in combination with the PI3K/Akt inhibitor wortmannin (100 nmol/L). In an additional protocol, ischaemia was followed by 2 h reperfusion for infarct size (IS) determination (tetrazolium staining). The total number of premature ventricular complexes over the whole period of ischaemia, episodes of ventricular tachycardia and its duration were significantly lower in the D-PC group than in the non-preconditioned controls (158 +/- 20, 2 +/- 0.6 and 4.6 +/- 1.8 s vs. 551 +/- 61, 11 +/- 2 and 42 +/- 8 s, respectively; p < 0.05), concomitant with a 62% reduction in the size of infarction. Wortmannin modified neither arrhythmogenesis nor IS in the non-preconditioned hearts. Bracketing of diazoxide with wortmannin did not reverse the antiarrhythmic protection, whereas the IS-limiting effect was blunted. The results indicate that in contrast with the positive role of PI3K/Akt in protection against lethal myocardial injury, its activity is not involved in suppression of ischaemia-induced arrhythmias conferred by mitoKATP opening in the rat heart.

Differential role of PI3K/Akt pathway in the infarct size limitation and antiarrhythmic protection in the rat heart.

Endogenous cardiac protection against prolonged ischemic insult can be achieved by repeated brief episodes of ischemia (hypoxia) or by cardiac adaptation to various stresses such as chronic hypoxia. Activation of phosphatidylinositol 3-kinase (PI3K)/Akt is involved in antiapoptotic effects, however, it is not clear whether it is required for overall heart salvage including protection against myocardial infarction and arrhythmias. We focussed on the potential common role of PI3K/Akt in anti-infarct protection, in the experimental settings of long-term adaptation to chronic intermittent hypobaric hypoxia (IHH; 8 h/day, 25-30 exposures, in vivo rats) and acute ischemic preconditioning (IP; Langendorff-perfused hearts). In addition, we explored the role of PI3K/Akt in susceptibility to ischemic ventricular arrhythmias. In normoxic open-chest rats, PI3K/Akt inhibitor LY294002 (LY; 0.3 mg/kg) given 5 min before test occlusion/reperfusion (I/R) did not affect infarct size (IS) normalized to the size of area at risk (AR). In hypoxic rats, LY partially attenuated IS-limiting effect of IHH (IS/AR 59.7 +/- 4.1% vs. 51.8 +/- 4.4% in the non-treated rats; p > 0.05) and increased IS/AR to its value in normoxic rats (64.9 +/- 5.1%). In the isolated hearts, LY (5 muM) applied 15 min prior to I/R completely abolished anti-infarct protection by IP (IS/AR 55.0 +/- 4.9% vs. 15.2 +/- 1.2% in the non-treated hearts and 42.0 +/- 5.5% in the non-preconditioned controls; p < 0.05). In the non-preconditioned hearts, PI3K/Akt inhibition did not modify IS/AR, on the other hand, it markedly suppressed arrhythmias. In the LY-treated isolated hearts, the total number of ventricular premature beats and the incidence of ventricular tachycardia (VT) was reduced from 518 +/- 71 and 100% in the controls to 155 +/- 15 and 12.5%, respectively (p < 0.05). Moreover, bracketing of IP with LY did not reverse antiarrhythmic effect of IP. These results suggest that activation of PI3K/Akt cascade plays a role in the IS-limiting mechanism in the rat heart, however, it is not involved in the mechanisms of antiarrhythmic protection.