Direct Rivaroxaban-Induced Factor Xa Inhibition Proves to be Cardioprotective in Rats

BACKGROUND: Acute myocardial infarction is a leading cause of death worldwide. Though highly beneficial, reperfusion of myocardium is associated with reperfusion injury. While indirect inhibition of Factor Xa has been shown to attenuate myocardial ischemia-reperfusion (I/R) injury, the underlying mechanism remains unclear. Our study sought to evaluate the effect of rivaroxaban (RIV), a direct inhibitor of Factor Xa, on myocardial I/R injury and determine its cellular targets. EXPERIMENTAL APPROACH: We used a rat model of 40-minutes coronary ligation followed by reperfusion. RIV (3 mg/Kg) was given per os 1 hour before reperfusion. Infarct size and myocardial proteic expression of survival pathways were assessed at 120 and 30 minutes of reperfusion, respectively. Plasmatic levels of P-selectin and von Willebrand factor were measured at 60 minutes of reperfusion. Cellular RIV effects were assessed using hypoxia-reoxygenation (H/R) models on human umbilical vein endothelial cells and on rat cardiomyoblasts (H9c2 cell line). KEY RESULTS: RIV decreased infarct size by 21% (42.9% vs. 54.2% in RIV-treated rats and controls respectively, p < 0.05) at blood concentrations similar to human therapeutic (387.7 ± 152.3 ng/mL) levels. RIV had no effect on H/R-induced modulation of endothelial phenotype, nor did it alter myocardial activation of RISK and SAFE pathways at 30 min after reperfusion. However, RIV exerted a cytoprotective effect on H9c2 cells submitted to H/R. CONCLUSION: RIV decreased myocardial I/R injury in rats at concentrations similar to human therapeutic ones. This protection was not associated with endothelial phenotype modulation but rather with potential direct cytoprotection on cardiomyocytes

Remote Ischemic Conditioning Influences Mitochondrial Dynamics

Remote ischemic preconditioning (RIPC) has emerged as an attractive strategy to protect the heart against ischemia-reperfusion (I/R) injury. The mechanisms by which remote ischemic conditioning (RIC) is protective are to date unknown, yet a well-accepted theory holds that the mitochondria play a central role. Mitochondria are dynamic organelles that undergo fusion and fission. Interventions that decrease mitochondrial fission or increase mitochondrial fusion have been associated with reduced I/R injury. However, whether RIPC influences mitochondrial dynamics or not has yet to be ascertained.We sought to determine the role played by mitochondrial dynamics in RIPC-induced cardioprotection. Male adult rats exposed in vivo to myocardial I/R were assigned to one of two groups, either undergoing 40 min of myocardial ischemia followed by 120 min of reperfusion (MI group) or four 5-min cycles of limb ischemia interspersed by 5 min of limb reperfusion, immediately prior to myocardial ischemia and 120 min of reperfusion (MI+RIPC group). After reperfusion, infarct size was assessed and myocardial tissue was analyzed by Western blot and electron microscopy. RIPC induced smaller infarct size (-28%), increased mitochondrial fusion protein OPA1, and preserved mitochondrial morphology. These findings suggest that mitochondrial dynamics play a role in the mechanisms of RIPC-induced cardioprotection

Microparticle release in remote ischemic conditioning mechanism

Remote ischemic conditioning (RCond) induced by short periods of ischemia and reperfusion of an organ or tissue before myocardial reperfusion is an attractive strategy of cardioprotection in the context of acute myocardial infarction. Nonetheless, its mechanism remains unknown. A humoral factor appears to be involved, although its identity is currently unknown. We hypothesized that the circulating microparticles (MPs) are the link between the remote tissue and the heart. MPs from rats and healthy humans undergoing RCond were characterized. In rats, RCond was induced by 10 min of limb ischemia. In humans, RCond was induced by three cycles of 5-min inflation and 5-min deflation of a blood-pressure cuff. In the second part of the study, rats underwent 40 min myocardial ischemia followed by 2 h reperfusion. Infarct size was measured and compared among three groups of rats: 1) myocardial infarction alone (MI) (n = 6); 2) MI + RCond started 20 min after coronary ligation (n = 6); and 3) MI + injection of RCond-derived rat MPs (MI + MPs) (n = 5). MPs from endothelial cells (CD54(+) and CD146(+) for rats and humans, respectively) and procoagulant MPs (Annexin V(+)) markedly increased after RCond, both in rats and humans. RCond reduced infarct size (24.4 ± 5.9% in MI + RCond vs. 54.6 ± 4.7% in MI alone; P < 0.01). Infarct size did not decrease in MI + MPs compared with MI alone (50.2 ± 6.4% vs. 54.6 ± 4.7%, not significantly different). RCond increased endothelium-derived and procoagulant MPs in both rats and humans. However, MP release did not appear to be a biological vector of RCond in our model

RISK and SAFE signaling pathway interactions in remote limb ischemic perconditioning in combination with local ischemic postconditioning

Local ischemic postconditioning (IPost) and remote ischemic perconditioning (RIPer) are promising methods to decrease ischemia–reperfusion (I/R) injury. We tested whether the use of the two procedures in combination led to an improvement in cardioprotection through a higher activation of survival signaling pathways. Rats exposed to myocardial I/R were allocated to one of the following four groups: Control, no intervention at myocardial reperfusion; IPost, three cycles of 10-s coronary artery occlusion followed by 10-s reperfusion applied at the onset of myocardial reperfusion; RIPer, 10-min limb ischemia followed by 10-min reperfusion initiated 20 min after coronary artery occlusion; IPost+RIPer, IPost and RIPer in combination. Infarct size was significantly reduced in both IPost and RIPer (34.25 ± 3.36 and 24.69 ± 6.02%, respectively) groups compared to Control (54.93 ± 6.46%, both p < 0.05). IPost+RIPer (infarct size = 18.04 ± 4.86%) was significantly more cardioprotective than IPost alone (p < 0.05). RISK pathway (Akt, ERK1/2, and GSK-3β) activation was enhanced in IPost, RIPer, and IPost+RIPer groups compared to Control. IPost+RIPer did not enhance RISK pathway activation as compared to IPost alone, but instead increased phospho-STAT-3 levels, highlighting the crucial role of the SAFE pathway. In IPost+RIPer, a SAFE inhibitor (AG490) abolished cardioprotection and blocked both Akt and GSK-3β phosphorylations, whereas RISK inhibitors (wortmannin or U0126) abolished cardioprotection and blocked STAT-3 phosphorylation. In our experimental model, the combination of IPost and RIPer improved cardioprotection through the recruitment of the SAFE pathway. Our findings also indicate that cross talk exists between the RISK and SAFE pathways

Myocardial reperfusion injury management: erythropoietin compared with postconditioning

Ischemic postconditioning (IPost) and erythropoietin (EPO) have been shown to attenuate myocardial reperfusion injury using similar signaling pathways. The aim of this study was to examine whether EPO is as effective as IPost in decreasing postischemic myocardial injury in both Langendorff-isolated-heart and in vivo ischemia-reperfusion rat models. Rat hearts were subjected to 25 min ischemia, followed by 30 min or 2 h of reperfusion in the isolated-heart study. Rats underwent 45 min ischemia, followed by 24 h of reperfusion in the in vivo study. In both studies, the control group (n = 12; ischemia-reperfusion only) was compared with IPost (n = 16; 3 cycles of 10 s reperfusion/10 s ischemia) and EPO (n = 12; 1,000 IU/kg) at the onset of reperfusion. The following resulted. First, in the isolated hearts, IPost or EPO significantly improved postischemic recovery of left ventricular developed pressure. EPO induced better left ventricular developed pressure than IPost at 30 min of reperfusion (73.18 ± 10.23 vs. 48.11 ± 7.92 mmHg, P < 0.05). After 2 h of reperfusion, the infarct size was significantly lower in EPO-treated hearts compared with IPost and control hearts (14.36 ± 0.60%, 19.11 ± 0.84%, and 36.21 ± 4.20% of the left ventricle, respectively; P < 0.05). GSK-3β phosphorylation, at 30 min of reperfusion, was significantly higher with EPO compared with IPost hearts. Phosphatidylinositol 3-kinase and ERK1/2 inhibitors abolished both EPO- and IPost-mediated cardioprotection. Second, in vivo, IPost and EPO induced an infarct size reduction compared with control (40.5 ± 3.6% and 28.9 ± 3.1%, respectively, vs. 53.7 ± 4.3% of the area at risk; P < 0.05). Again, EPO decreased significantly more infarct size and transmurality than IPost (P < 0.05). In conclusion, with the use of our protocols, EPO showed better protective effects than IPost against reperfusion injury through higher phosphorylation of GSK-3β

Role of hypoxia inducible factor-1α in remote limb ischemic preconditioning.

Remote ischemic preconditioning (RIPC) has emerged as a feasible and attractive therapeutic procedure for heart protection against ischemia/reperfusion (I/R) injury. However, its molecular mechanisms remain poorly understood. Hypoxia inducible factor-1α (HIF-1α) is a transcription factor that plays a key role in the cellular adaptation to hypoxia and ischemia. This study\u27s aim was to test whether RIPC-induced cardioprotection requires HIF-1α upregulation to be effective. In the first study, wild-type mice and mice heterozygous for HIF1a (gene encoding the HIF-1α protein) were subjected to RIPC immediately before myocardial infarction (MI). RIPC resulted in a robust HIF-1α activation in the limb and acute cardioprotection in wild-type mice. RIPC-induced cardioprotection was preserved in heterozygous mice, despite the low HIF-1α expression in their limbs. In the second study, the role of HIF-1α in RIPC was evaluated using cadmium (Cd), a pharmacological HIF-1α inhibitor. Rats were subjected to MI (MI group) or to RIPC immediately prior to MI (R-MI group). Cd was injected 18 0min before RIPC (Cd-R-MI group). RIPC induced robust HIF-1α activation in rat limbs and significantly reduced infarct size (IS). Despite Cd\u27s inhibition of HIF-1α activation, RIPC-induced cardioprotection was preserved in the Cd-R-MI group. RIPC applied immediately prior to MI increased HIF-1α expression and attenuated IS in rats and wild-type mice. However, RIPC-induced cardioprotection was preserved in partially HIF1a-deficient mice and in rats pretreated with Cd. When considered together, these results suggest that HIF-1α upregulation is unnecessary in acute RIPC

Diabetes mellitus abrogates erythropoietin-induced cardioprotection against ischemic-reperfusion injury by alteration of the RISK/GSK-3β signaling

Recent studies reported cardioprotective effects of erythropoietin (EPO) against ischemia–reperfusion (I/R) injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been reported to be impaired in diabetes and insulin resistance syndrome, we examined whether EPO-induced cardioprotection was maintained in rat models of type 1 diabetes and insulin resistance syndrome. Isolated hearts were obtained from three rat cohorts: healthy controls, streptozotocin (STZ)-induced diabetes, and high-fat diet (HFD)-induced insulin resistance syndrome. All hearts underwent 25 min ischemia and 30 min or 120 min reperfusion. They were assigned to receive either no intervention or a single dose of EPO at the onset of reperfusion. In hearts from healthy controls, EPO decreased infarct size (14.36 ± 0.60 and 36.22 ± 4.20% of left ventricle in EPO-treated and untreated hearts, respectively, p < 0.05) and increased phosphorylated forms of Akt, ERK1/2, and their downstream target GSK-3β. In hearts from STZ-induced diabetic rats, EPO did not decrease infarct size (32.05 ± 2.38 and 31.88 ± 1.87% in EPO-treated and untreated diabetic rat hearts, respectively, NS) nor did it increase phosphorylation of Akt, ERK1/2, and GSK-3β. In contrast, in hearts from HFD-induced insulin resistance rats, EPO decreased infarct size (18.66 ± 1.99 and 34.62 ± 3.41% in EPO-treated and untreated HFD rat hearts, respectively, p < 0.05) and increased phosphorylation of Akt, ERK1/2, and GSK-3β. Administration of GSK-3β inhibitor SB216763 was cardioprotective in healthy and diabetic hearts. STZ-induced diabetes abolished EPO-induced cardioprotection against I/R injury through a disruption of upstream signaling of GSK-3β. In conclusion, direct inhibition of GSK-3β may provide an alternative strategy to protect diabetic hearts against I/R injury

Endoplasmic reticulum stress pathway involvement in local and remote myocardial ischemic conditioning.

Remote ischemic perconditioning (RIPer) and local ischemic postconditioning (IPost) are promising methods to decrease ischemia-reperfusion injury. We tested whether these two methods were effective in reducing infarct size through activation of endoplasmic reticulum (ER) stress response, a potential survival pathway. Rats exposed to myocardial ischemia-reperfusion were allocated to one of six groups: control, no intervention at myocardial reperfusion; IPost, three cycles of 10-s coronary artery occlusion followed by 10-s reperfusion applied at the onset of myocardial reperfusion; RIPer, 10-min limb ischemia followed by 10-min reperfusion initiated during coronary artery occlusion; control + 4-PBA, injection of ER stress inhibitor 4-phenylbutyrate (4-PBA) 1 h before coronary occlusion; IPost + 4-PBA; and RIPer + 4-PBA. Infarct size was significantly reduced in IPost and RIPer groups (33.32% ± 3.65% and 21.86% ± 3.98%, respectively) compared with the control group (54.86% ± 6.01%, P < 0.05). Western blot analysis of GRP78 (glucose-regulated protein) level and cleaved activating transcription factor 6, two ER stress markers, demonstrated an enhancement of ER stress response in IPost group but not in RIPer group at 15-min reperfusion. Furthermore, 4-PBA abolished cardioprotection induced by IPost (infarct size 53.75 ± 3.49 vs. 33.32 ± 3.65%, P < 0.05) but not by RIPer (28.80 ± 10.45% vs. 21.86 ± 3.98%, not statistically significant). GRP78 and cleaved activating transcription factor 6 levels were no longer increased in IPost group after 4-PBA. These findings point to a role for ER stress response in cardioprotection against reperfusion injury in IPost but not RIPer, suggesting differences in cardioprotective mechanisms between local and remote conditioning

The Synthetic Pentasaccharide Fondaparinux Attenuates Myocardial Ischemia-Reperfusion Injury in Rats Via STAT-3

Acute myocardial infarction is a leading cause of mortality and morbidity worldwide. Although essential for successful recovery, myocardium reperfusion is associated with reperfusion injury. Two major cell survival signaling cascades are known to be protective against ischemia-reperfusion (I/R) injury: the reperfusion injury salvage kinase, including Akt, extracellular signal–regulated kinase 1/2, and the downstream target GSK-3β, and the survivor activating factor enhancement, which involves STAT-3. Pharmacologic inhibition of factor Xa has been shown to attenuate I/R injury, but the cellular mechanism is poorly understood. Our aim was to determine the role of whole blood in fondaparinux (FDX)-induced cardioprotection and the involvement of reperfusion injury salvage kinase and survivor activating factor enhancement pathways. We investigated FDX ability to prevent in vivo I/R injury using a transient coronary ligation rat model and ex vivo using a model of crystalloid-perfused isolated rat heart. In both models, infarct size was assessed after 120 min of reperfusion. Myocardial tissues were collected after 15 and 30 min of reperfusion for Western blot analysis. In vivo, FDX decreased infarct size by 29% and induced significant STAT-3 and GSK-3β phosphorylation in comparison to controls. Adding AG490, an inhibitor of JAK/STAT pathway, before I/R, prevented STAT-3 phosphorylation and abolished FDX-induced cardioprotection. On the contrary, FDX did not have an effect on infarct size or hemodynamic parameters in the isolated-heart model. Fondaparinux decreased I/R injury in vivo, but not in a crystalloid-perfused isolated heart. Under our experimental conditions, FDX required whole blood to be protective, and this beneficial effect was mediated through STAT-3 phosphorylation

Speckle tracking imaging improves in vivo assessment of EPO-induced myocardial salvage early after ischemia-reperfusion in rats

A noninvasive assessment of infarct size and transmural extension of myocardial infarction (TEMI) is fundamental in experimental models of ischemia-reperfusion. Conventional echocardiography parameters are limited in this purpose. This study was designed to examine whether speckle tracking imaging can be used in a rat model of ischemia-reperfusion to accurately detect the reduction of infarct size and TEMI induced by erythropoietin (EPO) as early as 24 h after reperfusion. Rats were randomly assigned to one of three groups: myocardial infarction (MI)-control group, 45 min ischemia followed by 24 h of reperfusion; MI-EPO group, similar surgery with a single bolus of EPO administered at the onset of reperfusion; and sham-operated group. Short-axis two-dimensional echocardiography was performed after reperfusion. Global radial (GSr) and circumferential (GScir) strains were compared with infarct size and TEMI assessed after triphenyltetrazolium chloride staining. As a result, ejection fraction, shortening fraction, GSr, and GScir significantly correlated to infarct size, whereas only GSr and GScir significantly correlated to TEMI. EPO significantly decreased infarct size (30.8 ± 3.5 vs. 56.2 ± 5.7% in MI-control, P < 0.001) and TEMI (0.37 ± 0.05 vs. 0.77 ± 0.05 in MI-control, P < 0.001). None of the conventional echocardiography parameters was significantly different between the MI-EPO and MI-control groups, whereas GSr was significantly higher in the MI-EPO group (29.1 ± 4.7 vs. 16.4 ± 3.3% in MI-control; P < 0.05). Furthermore, GScir and GSr appeared to be the best parameters to identify a TEMI >0.75 24 h after reperfusion. In conclusion, these findings demonstrate the usefulness of speckle tracking imaging in the early evaluation of a cardioprotective strategy in a rat model of ischemia-reperfusion