Insulin Improves Cardiomyocyte Contractile Function Through Enhancement Of SERCA2a Activity In Simulated Ischemia/reperfusion

BackgroundReduced contractility and relaxation have been found in rat hearts after ischemia/reperfusion (I/R), accompanied by reduced peak in Ca²⁺ transients and prolonged Ca²⁺ transients decay. Cardiac sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a) regulates Ca2+ in cytoplasm by alteration of SR Ca²⁺ intake. Reduced expression and activity of SERCA2a are key features of intracellular Ca²⁺overload and cardiac myocyte dysfunction.Our previous study showed that insulin improved cardiac myocyte function recovery following I/R period in vitro experiments by activating the phosphatidylinositol 3-kinase-Akt (PI3Kinase-Akt) pathway, that is, the survival signaling pathway. And Akt activation was the critical step of insulin-induced improvement. However, the downstream mechanisms of I/R myocyte functional recovery are largely elusive. Some studies showed that in cardiomyocytes isolated from the left ventricle of patients with end-stage HF, SERCA2a overexpression could restore normal Ca2+ cycling. In addition, transgenic overexpression of protein kinase Akt in mice enhanced myocyte contractility and relaxation through acceleration of intracellular Ca²⁺ transients . Nevertheless, to date, a direct link between the alteration of SERCA2a activity and expression in I/R myocardium and the insulin-induced survival pathway and subsequent improvement of I/R myocyte functional recovery has not been established.Aims1. To investigate the effect of insulin on expression and activity of SERCA2a in stimulated rat I/R myocytes.2. To elucidate the role of SERCA2a in the insulin-induced improvement of contractile function and intracellular Ca²⁺ transients in I/R cardiomyocytes and the underlying mechanism.Methods1. Ventricular myocytes were enzymatically isolated from adult Sprague-Dawley rats. Isolated rat hearts and myocytes were randomly divided into four groups: (1) Control; (2) I/R; (3) I/R plus insulin (10-7 mol/L); (4) I/R plus insulin plus Akt inhibitor (AI).2. Simulated I/R modle was induced by perfusing cells with chemical anoxic solution for 15 min followed by reperfusion for 30 min.3. The mechanical contraction of ventricular myocytes was assessed by a video-based motion edge-detection system. Myocyte Ca²⁺ transients were measured with the same system.4. To examine Akt, pAkt, SERCA2a, PLB, which is the natural profiling of SERCA2a, and pPLB expression in cardiomyocytes, Western Blot analysis was performed.5. The activity of Ca²⁺-ATPase was determined with a kit by measuring the inorganic phosphate (Pi) liberated from ATP hydrolysis.Results1. Insulin improved myocyte contractile and relaxation function in stimulated I/R myocytes.Reperfusion with insulin (10-7 mol/L) significantly improved peak twitch amplitude (PTA%baseline) (20.7±0.7 % of I/R+Ins group vs. 14.5±0.7 % of I/R group; P<0.05, n = 20), and shortened time to peak shortening (TPS), time to 70 % relengthening (TR70), and the maximal velocities of shortening/relengthening (±dL/dt) (322±12, 302±7μm·s^-1 of I/R+Ins group vs. 279±29, 241±10μm·s^-1 of I/R group; P<0.05, n = 20) were increased compared with those in cells reperfused with vehicle. These data showed that insulin significantly improved myocyte contractile and relaxation function after I/R.2. Insulin increased intracellular Ca²⁺ transients in stimulated I/R myocytes.Contracted with the I/R group, reperused insulin increased calcium transients ratio as evidenced by the increased Ca²⁺ fluorescence ratio (0.36±0.04 of I/R+Ins group vs. 0.32±0.03 of the I/R group; P<0.05, n = 20). And in I/R+Ins group, time to peak Ca²⁺ (61.2±2.0 ms of I/R+Ins group vs. 77.5±3.5 ms of the I/R group; P<0.01, n = 20) and to 50% diastolic Ca²⁺ (206±9 ms of I/R+Ins group vs. 278±8 ms of the I/R group; P<0.01, n = 20) were deduced. These data gave the evidence that insulin could increase myocyte Ca²⁺ transients after I/R.3. Insulin augmented phosphorylative activation of Akt and PLB in stimulated I/R myocytes, as well as activity of SERCA2a.Although the I/R procedure made no change in Akt and PLB expression, treatment with insulin resulted in increasement in Akt phosphorylation (P<0.01 vs. I/R group, n = 5), and pPLB expression (P<0.01 vs. I/R group, n = 5) which removed the inhibition of SERCA2a activity was increased, either.In our study, there was no augment in signal intensity of the SERCA2a in rat cardiomyocytes of I/R+Ins group contrasted with I/R group (P>0.05,n = 20). But, it was seen that SERCA2a activity was significantly decreased in I/R myocytes, and treatment with insulin at the onset of reperfusion significantly increased the SERCA2a activity in I/R+Ins myocytes (7.8±0.4 mol Pi·mg protein^-1·h^-1 of I/R+Ins group vs. 6.8±0.6 mmol Pi·mg protein^-1·h^-1 of I/R group, P<0.05, n = 8). These results suggested that the insulin-induced improvement of I/R myocytes function by activating Akt signaling pathway accompanied with its effects on PLB phosphorylation and SERCA2a activity.4. AI could abolished the contractile effect, the augmented intracellular calcium transients, the Akt and PLB phosphorylation and the augmentation of SERCA2a activity induced by insulin.Pre-treatment with AI not only markedly decreased the Akt phosphorylation (I/R+Ins+AI vs. I/R+Ins, P<0.01, n = 5) and pPLB (I/R+Ins+AI vs. I/R+Ins, P<0.05, n = 5) by insulin, but also eliminated the augmentation of SERCA2a activity in the insulin-treated I/R myocytes (I/R+Ins+AI vs. I/R+Ins, P<0.05, n = 8).In addition, AI treatment also almost completely abolished the contractile effect contrasted with I/R group (P<0.05 vs. I/R+Ins, n = 20), and the augmented intracellular calcium transients induced by insulin synchronously (P<0.01 vs. I/R+Ins, n = 20).These results provided direct evidence that insulin removed inhibition of PLB to SERCA2a activity, which is at least partly by enhanced Akt activation, and regulated intracellular calcium transients and improved cardiac contractile function in I/R myocytes.Conclusions1. Reperfusion of insulin exerts stimulated effect to contractive function and the level of intracellular calcium transients in I/R cardiomyocytes.2. Reperfusion of insulin increases activity of I/R cardiomyocyte SERCA2a, which plays as a downstream molecule and involves in the insulin-induced improvements of cardiac contractile function and intracellular calcium transients in I/R cardiomyocyte by adjusting intracellular Ca²⁺.