Cardiomyocyte Protective Effect Of Thyroid Hormone During Hypoxia/Reoxygenation Injury Through Activating Of PI3K/Akt Signaling

Objective:Ischemic heart disease leads to the highest mortality rate in the world,and the recovery of blood flow in the ischemic area will lead to ischemia/reperfusion injury,a serious complication,leading to myocardial mitochondrial injury,calcium overload and severe myocardial cell apoptosis,resulting in irreversible damage and dysfunction of the heart.Thyroid hormone can promote cardiac function recovery after cardiac injury.However,whether T3 could protect myocardium from I/R injury,and the possible molecular mechanism is still unclear.The present study was to investigate the effect of thyroid hormone T3 on neonatal mouse cardiomyocytes under hypoxia/reoxygenation（H/R）condition,and the role of PI3K/Akt signaling pathway.Methods:Cardiomyocytes were cultured from 1-2 days neonatal mice.Different concentration of T3（0,20,40,and 80μM）was used for 24 h pretreatment,respectively.Then,cells were treated with 4 h hypoxia and 3 h reoxygenation for H/R injury model,the effect of T3 on cardiomyocytes was studied.The cultured cardiomyocytes were divided into five groups,including control group,H/R group,H/R+T3 group,H/R+T3+LY294002 group,H/R+LY294002 group.After 24 h of T3 pretreatment,cells were treated with H/R injury.LY294002,a PI3K/Akt signaling blocker,was pretreated 24 h before cardiomyocytes under H/R injury.Apoptosis rates of cardiomyocytes were detected by Annexin-V FITC/PI double staining and flow cytometry,mitochondrial membrane potential was detected by JC-1 staining with laser scanning confocal microscope.L-type calcium channel current density was detected by single-cell patch clamp technique.The expression of apoptotic protein（Bax,Bcl-2,caspase-3）,PI3K/Akt signal pathway and SERCA2a,NCX1 were detected by Western blot.Results:Compared with control group,T3 pretreatment could reduce cell death caused by hypoxia/reoxygenation injury,and 60μM T3 significantly increased the viability of cardiomyocytes,while high concentration of T3（80μM）showed cell cytotoxicity and decreased cell viability.Compared with control group,H/R injury increased the expression level of caspase-3 and Bax protein,and significantly decreased the expression of Bcl-2 protein in cardiomyocytes.Moreover,the cell apoptosis rate was significantly enhanced（40.47%vs 7.61%,P<0.05）.However,T3 pretreatment reduced cardiomyocytes apoptosis induced by H/R injury（28.41%vs 40.47%,P<0.05）,and T3 pretreatment also enhanced the expression of bcl-2 and reduced Bax and caspase-3 expression.While,the apoptosis rate of cardiomyocytes was significantly increased with LY294002 pretreatment（43.66%vs.28.41%,P<0.05）,the expression of bcl-2 was decreased and the Bax and caspase-3 protein expression were significantly increased（P<0.05）.Compared with control group,H/R decreased the mitochondrial membrane potential of cardiomyocytes,the green/red fluorescence ratio was significantly increased（P<0.05）.T3 pretreatment significantly reduced this decrease under H/R injury（P<0.05）.When LY294002 was added,the mitochondrial green/red fluorescence ratio was markedly increased and the mitochondrial membrane potential was significantly decreased（P<0.05）.Compared with control group,H/R injury decreased L-type Ca²⁺channel（LTCC）current density（6.97±0.485 vs 14.62±1.357 p A/p F,n=10,P<0.05）,with a upward shift of I-V curve.The activation curve of LTCC had a left shift,and the activation process was accelerated,while the inactivation curve had a right shift,the inactivation process was prolonged.However,T3 pretreatment increased the current density of LTCCunder H/R injury（10.66±0.719 vs 6.97±0.485 p A/p F,n=10,P<0.05）,and diminished the left-shift of activation curve and right-shift of the inactivation curve.While,the current density of L-type Ca²⁺channel was significantly decreased（3.264±0.24 p A/p F,n=9,P<0.05）with LY294002 pretreatment,and the activation curve had a left shift and the inactivation curve was shifted to the right.Compared with control group,hypoxia/reoxygenation injury increased the expression of sodium-calcium exchange protein（NCX1）and decreased the expression of SERCA2a protein（P<0.05）.However,pretreatment with T3 significantly reduced the expression of NCX1 protein and increased the expression of SERCA2a（P<0.05）.When LY294002 was added,this effect T3 significantly inhibited,the expression of cardiomyocyte sodium-calcium exchange protein（NCX1）was significantly increased and the expression of SERCA2a protein was obviously decreased（P<0.05）.Compared with the control group,H/R injury increased the phosphorylation of PI3K（P<0.05）,while the expression of phosphorylated Akt had no significant difference;T3 preconditioning significantly increased the expression of p-PI3K and p-Akt（P<0.05）.When LY294002 was added,the effect of T3 was significantly inhibited,and the expressions of p-PI3K and p-Akt were significantly decreased（P<0.05）.Conclusion:T3 pretreatment could increase the viability rate of cardiomyocytes under H/R injury,and inhibit apoptosis of cardiomyocytes by reducing the expression of apoptotic proteins caspase-3 and Bax.T3 pretreatment could diminished the reduce of mitochondrial membrane potential in H/R-induced cardiomyocytes,and protected L-type Ca²⁺channel by increasing the L-type Ca²⁺channel of cardiomyocytes induced by H/R injury.T3pretreatment also reduced the increased NCX1 expression and increased the expression of SERCA2a protein of cardiomyocytes under H/R condition,reduced intracellular calcium overload and protect cardiomyocytes.T3 can activate the IGF-1-mediated PI3K/Akt signaling pathway,and LY294002could inhibit the protective effect of T3 on myocardial cells,which indicated that T3could protect cardiomyocytes against hypoxia/reoxygenation-induced injury through activating of PI3K/Akt signaling pathway.