Nitrite circumvents platelet resistance to nitric oxide in patients with heart failure preserved ejection fraction and chronic atrial fibrillation

Aims: Heart failure (HF) is a pro-thrombotic state. Both platelet and vascular responses to nitric oxide (NO) donors are impaired in HF patients with reduced ejection fraction (HFrEF) compared to healthy volunteers (HV) due to scavenging of NO, and possibly also reduced activity of the principal NO sensor, soluble guanylate cyclase (sGC), limiting the therapeutic potential of NO donors as anti-aggregatory agents. Previous studies have shown that nitrite inhibits platelet activation presumptively after its reduction to NO, but the mechanism(s) involved remain poorly characterized. Our aim was to compare the effects of nitrite on platelet function in HV vs. HF patients with preserved ejection fraction (HFpEF) and chronic atrial fibrillation (HFpEF-AF), vs. patients with chronic AF without HF, and to assess whether these effects occur independent of the interaction with other formed elements of blood. Methods and Results: Platelet responses to nitrite and the NO donor sodium nitroprusside (SNP) were compared in age-matched HV controls (n = 12), HFpEF-AF patients (n = 29) and chronic AF patients (n = 8). Anti-aggregatory effects of nitrite in the presence of NO scavengers/sGC inhibitor were determined and vasodilator-stimulated phosphoprotein (VASP) phosphorylation was assessed using Western blotting. In HV and chronic AF, both nitrite and SNP inhibited platelet aggregation in a concentration-dependent manner. Inhibition of platelet aggregation by the NO donor SNP was impaired in HFpEF-AF patients compared to healthy and chronic AF individuals, but there was no impairment of the anti-aggregatory effects of nitrite. Nitrite circumvented platelet NO resistance independently of other blood cells by directly activating sGC and phosphorylating VASP. Conclusion: We here show for the first time that HFpEF-AF (but not chronic AF without HF) is associated with marked impairment of platelet NO responses due to sGC dysfunction and nitrite circumvents the “platelet NO resistance” phenomenon in human HFpEF, at least partly, by acting as a direct sGC activator independent of NO

Overexpression and pre-treatment of recombinant human Secretory Leukocyte Protease Inhibitor (rhSLPI) reduces an in vitro ischemia/reperfusion injury in rat cardiac myoblast (H9c2) cell

One of the major causes of cardiac cell death during myocardial ischemia is the oversecretion of protease enzymes surrounding the ischemic tissue. Therefore, inhibition of the protease activity could be an alternative strategy for preventing the expansion of the injured area. In the present study, we investigated the effects of Secretory Leukocyte Protease Inhibitor (SLPI), by means of overexpression and treatment of recombinant human SLPI (rhSLPI) in an in vitro model. Rat cardiac myoblast (H9c2) cells overexpressing rhSLPI were generated by gene delivery using pCMV2-SLPI-HA plasmid. The rhSLPI-H9c2 cells, mock transfected cells, and wild-type (WT) control were subjected to simulated ischemia/reperfusion (sI/R). Moreover, the treatment of rhSLPI in H9c2 cells was also performed under sI/R conditions. The results showed that overexpression of rhSLPI in H9c2 cells significantly reduced sI/R-induced cell death and injury, intracellular ROS level, and increased Akt phosphorylation, when compared to WT and mock transfection (p <0.05). Treatment of rhSLPI prior to sI/R reduced cardiac cell death and injury, and intra-cellular ROS level. In addition, 400 ng/ml rhSLPI treatment, prior to sI, significantly inhibited p38 MAPK phosphorylation and rhSLPI at 400–1000 ng/ml could increase Akt phosphorylation

Post-Ischemic Treatment of Recombinant Human Secretory Leukocyte Protease Inhibitor (rhSLPI) Reduced Myocardial Ischemia/Reperfusion Injury

International audienceMyocardial ischemia/reperfusion (I/R) injury is a major cause of mortality and morbidity worldwide. Among factors contributing to I/R injury, proteolytic enzymes could also cause cellular injury, expand the injured area and induce inflammation, which then lead to cardiac dysfunction. Therefore, protease inhibition seems to provide therapeutic benefits. Previous studies showed the cardioprotective effect of secretory leukocyte protease inhibitor (SLPI) against myocardial I/R injury. However, the effect of a post-ischemic treatment with SLPI in an in vivo I/R model has never been investigated. In the present study, recombinant human (rh) SLPI (rhSLPI) was systemically injected during coronary artery occlusion or at the onset of reperfusion. The results show that post-ischemic treatment with rhSLPI could significantly reduce infarct size, Lactate Dehydrogenase (LDH) and Creatine kinase-MB (CK-MB) activity, inflammatory cytokines and protein carbonyl levels, as well as improving cardiac function. The cardioprotective effect of rhSLPI is associated with the attenuation of p38 MAPK phosphorylation, Bax, caspase-3 and -8 protein levels and enhancement of pro-survival kinase Akt and ERK1/2 phosphorylation. In summary, this is the first report showing the cardioprotective effects against myocardial I/R injury of post-ischemic treatments with rhSLPI in vivo. Thus, these results suggest that SLPI could be used as a novel therapeutic strategy to reduce myocardial I/R injury

Combination of metformin and p38 MAPK inhibitor, SB203580, reduced myocardial ischemia/reperfusion injury in non‑obese type 2 diabetic Goto‑Kakizaki rats

International audienceDiabetic cardiomyopathy, especially myocardial ischemia reperfusion (I/R) injury, is a major cause of morbidity and mortality in type 2 diabetic patients. The increasing of basal p38 MAP Kinase (p38 MAPK) activation is a major factor that aggravates cardiac death on diabetic cardiomyopathy. In addition, metformin also shows cardio-protective effects on myocardial ischemia/reperfusion injury. In this study, we investigated the effect of the combination between metformin and p38 MAPK inhibitor (SB203580) in diabetic rats subjected to I/R injury. H9c2 cells were induced into a hyperglycemic condition and treated with metformin, SB203580 or the combination of metformin and SB203580. In addition, cells in both the presence and absence of drug treatment were subjected to simulated ischemia/reperfusion injury. Cell viability and cellular reactive oxygen species (ROS) were determined. Moreover, the Goto-Kakizaki (GK) rats were treated with metformin, SB203580, and the combination of metformin and SB203580 for 4 weeks. Diabetic parameters and cardiac functions were assessed. Finally, rat hearts were induced ischemia/reperfusion injury for the purpose of infarct size analysis and determination of signal transduction. A high-glucose condition did not reduce cell viability but significantly increased ROS production and significantly decreased cell viability after induced sI/R. Treatment using drugs was shown to reduce ROS generation and cardiac cell death. The GK rats displayed diabetic phenotype by increasing diabetic parameters and these parameters were significantly decreased when treated with drugs. Treatment with metformin or SB203580 could significantly reduce the infarct size. Interestingly, the combination of metformin and SB203580 could enhance cardio-protective ability. Myocardial I/R injury significantly increased p38 MAPK phosphorylation, Bax/Bcl-2 ratio and caspase-3 level. Treatment with drugs significantly decreased the p38 MAPK phosphorylation, Bax/Bcl-2 ratio, caspase-3 level and increased Akt phosphorylation. In conclusion, using the combination of metformin and SB203580 shows positive cardio-protective effects on diabetic ischemic cardiomyopathy