Linagliptin Improved Myocardial Function Recovery In Rat Hearts Suffered From Hypothermia Preservation

BackgroundCardiac transplantation is regarded as the primary therapy for end-stage heart diseases.Reduction of cardiac hypothermic preservation-induced injury has become more important in improvement of clinical heart transplantation practice.Linagliptin is a high selective dipeptidyl peptidase 4(DPP-4)inhibitor.It has been reported that linagliptin can inhibit DPP-4,reduce the catabolism of glucagon-like peptide 1 and gastric inhibitory peptides,and lower blood sugar levels.Since its potential effect on improving hyperglycemia condition and protecting beta cells,linagliptin is widely used as a new antidiabetic medicine in clinical practice.Recent studies have shown that DPP-4 inhibitors also play an important role in cardiovascular protection.DPP-4 inhibitors could reduce oxidative stress,improve heart rate variability and left ventricular function in chronic myocardial infarction rat hearts.DPP-4 inhibitors can also protect cardiovascular system by reducing macrophages infiltration and decreasing inflammatory cytokines CDllb and CDllc.So we hypothesize that supplement of Celsior solution with linagliptin might prolong the hypothermic preservation period,reduce the hypothermic preservation-induced cardiac dysfunction.ObjectivesThe aim of present study is to investigate the effect of linagliptin on cardiac function recovery after hypothermic preservation,and to explore its underlying mechanism.Methods(1)Langendorff perfusion model of isolated rat hearts.SD rat hearts were isolated and mounted onto the Langendorff perfusion system.Krebs-Henseleit solution(37?,saturation with 95%O2 and 5%CO2)were retrogradely perfused from the aortic root at constant pressure(76mmHg).(2)Establishment of rat heart hypothermic preservation model.Rat hearts were preserved in 4 ? Celsior solution for 9 h followed by 60 min of reperfusion with 37?Krebs-Henseleit solution.(3)Evaluation of cardiac function.Cardiac function indexes such as left ventricular end-diastolic pressure(LVEDP),left ventricular developed pressure(LVDP),Maximal systolic velocity of left ventricular pressure(+dP/dtmax),Maximal diastolic velocity of left ventricular pressure(-dP/dtmax,heart rate(HR)were recorded using MedLab biological signal acquisition and processing system.(4)Western blotting.Total and mitochondrial protein was extracted from heart tissue.Expression of Drp1,Drpl S616,CaMK II,p-CaMK II,RhoA,mitofusinl(Mfnl)and mitofusin2(Mfnl)were analyzed by western blotting.(5)Measurement of reactive oxygen species(ROS).The myocardial ROS level was measured by using DCFH-DA method in accordance with the manufacturer's protocol.(6)Determination of Lipid oxidation.Content of malondialdehyde(MDA)was detected as the oxidative metabolites of lipid.(7)Measurement of manganese superoxide dismutase(MnSOD)activity.The cardiac MnSOD activity was measured using WST-8 method in accordance with the manufacturer's protocol.(8)Evaluation of cardiac morphology.Left ventricle myofibril and mitochondrial morphology in rat hearts was observed by using Transmission electron microscope.Results(1)After hypothermic preservation for 9 h,cardiac function was evaluated at 60 min of reperfusion.LVEDP increased,LVDP and ±dP/dtmax were decreased.Compared with Celsior group,supplement of Celsior solution with linagliptin(0.50,0.75 nM)could significantly prevent hypothermic preservation-induced increase in LVEDP and decrease in LVDP and ± dP/dtmax.Linagliptin(0.25,0.50,or 0.75 nM)could improve hypothermic preservation-induced coronary flow decline.(2)The expression of NOX2 in the ventricle was significantly increased after 9 h of preservation and 60 min of reperfusion,which was inhibited by supplement of inagliptin(0.5 and 0.75 nM).Compared with Celsior group,linagliptin could also reduce ROS level and MDA content in cardium.(3)Although the total Drpl expression in myocardium did not change,the level of p-drpl S616 was significantly increased.Hypothermic preservation could not influence the expression of Mfnl and Mfn2 protein either.(4)Compared with control group,CaMK II and RhoA protein expression didn't change in hypothermic preserved rat hearts,but the level of p-CaMK ? increased.(5)Linagliptin and NOX2 inhibitor phox-I2 could significantly inhibit the hypothermic preservation-induced overexpression of p-CaMK ?,p-Drpl S616 and mitochondrial Drp1.Meanwhile,preservation-induced overexpression of p-Drpl S616 and mitochondrial Drp1 could also inhibited by CaMK II inhibitor KN-93.(6)Linagliptin could also significantly reduce left ventricle myofibril disarrangement and mitochondrial fragmentation caused by hypothermic preservation.ConclusionLinagliptin supplement could promote cardiac function recovery in hypothermic preservated rat hearts.The mechanism might be involved in reduction of hypothermic preservation-induced NOX2 overexpression,and inhibition of Drp1 S616 phosphorylation and mitochondrial fission.