Crocetin protects cardiomyocytes against hypoxia/reoxygenation injury by attenuating Drp1-mediated mitochondrial fission via PGC-1α

BACKGROUND Saffron(Crocus sativus L.)has been traditionally used as food,spice,and medicine.Crocetin(CRT),as main bioactive component of saffron,has accumulated pieces of beneficial evidence on myocardial ischemia/reperfusion(I/R)injury.However,the mechanisms are poorly explored.This study aims to investigate the effects of CRT on H9c2 cells under hypoxia/reoxygenation(H/R)and elucidated the possible underlying mechanism.METHODS H/R attack was performed on H9c2 cells.Cell counting kit-8 was used to detect the cell viability.Cell samples and culture supernatants were evaluated via commercial kits to measure the superoxide dismutase(SOD)activity,malondialdehyde(MDA)content,and cellular adenosine triphosphate(ATP)content.Various fluorescent probes were used to detect cell apoptosis,intracellular and mitochondrial reactive oxygen species(ROS)content,mitochondrial morphology,mitochondrial membrane potential(MMP),and mitochondrial permeability transition pore(mPTP)opening.Proteins were evaluated via Western Blot.RESULTS H/R exposure severely reduced cell viability and increased LDH leakage.Peroxisome proliferator-activated receptorγcoactivator-1α(PGC-1α)suppression and dynamin-related protein 1(Drp1)activation were coincided with excessive mitochondrial fission,mitochondrial permeability transition pore(mPTP)opening and mitochondrial membrane potential(MMP)collapse in H9c2 cells treated with H/R.Mitochondria fragmentation under H/R injury induced ROS over-production,oxidative stress,and cell apoptosis.Notably,CRT treatment significantly prevented mitochondrial fission,mPTP opening,MMP loss,and cell apoptosis.Moreover,CRT sufficiently activated PGC-1α and inactivated Drp1.Interestingly,mitochondrial fission inhibition with mdivi-1 similarly suppressed mitochondrial dysfunction,oxidative stress and cell apoptosis.However,silencing PGC-1α with small interfering RNA(siRNA)abolished the beneficial effects of CRT on H9c2 cells under H/R injury,accompanied with increased Drp1 and p-Drp1ser616 levels.Furthermore,over-expression of PGC-1αwith adenovirus transfection replicated the beneficial effects of CRT on H9c2 cells.CONCLUSIONS Our study identified PGC-1α as a master regulator in H/R-injured H9c2 cells via Drp1-mediated mitochondrial fission.We also presented the evidence that PGC-1α might be a novel target against cardiomyocyte H/R injury.Our data revealed the role of CRT in regulating PGC-1α/Drp1/mitochondrial fission process in H9c2 cells under the burden of H/R attack,and we suggested that modulation of PGC-1α level may provide a therapeutic target for treating cardiac I/R injury.

Levosimendan versus dobutamine in critically ill patients: a meta-analysis of randomized controlled trials

Objective: To evaluate the clinical efficacy of levosimendan versus dobutamine in critically ill patients requiring inotropic support. Methods: Clinical trials were searched in PubMed, EMBASE, and the Cochrane Central Registry of Clinical Trials, as well as Web of Science. Studies were included if they compared levosimendan with dobutamine in critically ill patients requiring inotropic support, and provided at least one outcome of interest. Outcomes of interest included mortality, incidence of hypotension, supraventricular arrhythmias, and ventricular arrhythmias. Results: Data from a total of 3052 patients from 22 randomized controlled trials (RCTs) were included in the analysis. Overall analysis showed that the use of levosimendan was associated with a significant reduction in mortality (269 of 1373 [19.6%] in the levosimendan group, versus 328 of 1278 [25.7%] in the dobutamine group, risk ratio (RR)=0.81, 95% confidence interval (CI) 0.70-0.92, P for effect=0.002). Subgroup analysis indicated that the benefit from levosimendan could be found in the subpopulations of cardiac surgery, ischemic heart failure, and concomitant β-blocker therapy in comparison with dobutamine. There was no significant difference in the incidence of hypotension, supraventricular arrhythmias, or ventricular arrhythmias between the two drugs. Conclusions: In contrast with dobutamine, levosimendan is associated with a significant improvement in mortality in critically ill patients requiring inotropic support. Patients having cardiac surgery, with ischemic heart failure, and receiving concomitant β-blocker therapy may benefit from levosimendan. More RCTs are required to address the questions about no positive outcomes in the subpopulation in a cardiology setting, and to confirm the advantages in long-term prognosis.

Angiopoietin-1 preconditioning enhances survival and functional recovery of mesenchymal stem cell transplantation

Objective： Mesenchymal stem cell （MSC） transplantation is a promising therapy for ischemic heart diseases. However, poor cell survival after transplantation greatly limits the therapeutic efficacy of MSCs. The purpose of this study was to investigate the protective effect of angiopoietin-1 （Angl） preconditioning on MSC survival and sub- sequent heart function improvement after transplantation. Methods： MSCs were cultured with or without 50 ng/ml Angl in complete medium for 24 h prior to experiments on cell survival and transplantation. 3-（4,5- Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） and Hoechst staining were applied to evaluate MSC survival after serum deprivation in vitro, while cell survival in vivo was detected by terminal deoxynucleotidyl trans- ferase biotin-dUPT nick end labeling （TUNEL） assay 24 and 72 h after transplantation. Heart function and infarct size were measured four weeks later by small animal echocardiography and Masson＇s trichrome staining, respectively. Results： Angl preconditioning induced Akt phosphorylation and increased expression of Bcl-2 and the ratio of Bcl-2/Bax. In comparison with non-preconditioned MSCs, Angl-preconditioned cell survival was significantly in- creased while the apoptotic rate decreased in vitro. However, the PI3K/Akt pathway inhibitor, LY294002, abrogated the protective effect of Angl preconditioning. After transplantation, the Angl-preconditioned-MSC group showed a lower death rate, smaller infarct size, and better heart functional recovery compared to the non-preconditioned-MSC group. Conclusions： Angl preconditioning enhances MSC survival, contributing to further improvement of heart function.