Effects Of Simvastatin On Sodium-hydrogen Exchanger In Rat Cardiomyocytes

Part oneEffects of simvastatin on sodium-hydrogen exchanger in ratcardiomycytesObjective:Simvastatin is not only due to prevent chronic cardiovascular diseases, but also produce the protection for ischemia-reperfusion induced cardiac injury in diabetic mice. Na+/H+ exchanger activation is the major factor for myocardial ischemia-reperfusion injury. The earlier research indicated that simvastatin decreased the pH value of cancer cells. Our recent research also showed that simvastatin can quickly inhibited the elevation of intracellular pH and Ca2+ concentration induced by hydrogen peroxide. Pre-treatment of simvastatin can also prevented the elevation of intracellular pH and Ca2+ concentration induced by hydrogen peroxide, and cardiomyocyte apoptosis. Therefore, we hypothesised that simvastatin play mycardial protective effects via inhibiting Na+/H+ exchanger activity.Methods:The study was performed with primary cultured neonatal rat cardiomyocytes. In all cases, intracellular acidosis was induced in cardiomyocytes bathed in bicarbonate-free Kreb's solution by transient 3 min exposure to NH4Cl (25 mM), and then washouted NH4Cl with Na+-free Kreb's solution and bathed for 3 min, the extracellular buffer contains NH4+ and NH3 in an equilibrium that is essentially recapitulated in the cell interior. When the extracellular medium is changed to a buffer lacking NH4Cl, intracellular NH3 diffuses rapidly out of the cell, causing the increase of intracellular H+. After the recovery of Kreb's solution (with the introduction of Na+), it can activate Na+/H+ exchanger and restore the intracellular pH; Intracellular pH was measured using microplate reader of multi-wavelength measurement system; As the index of NHE activity , the rate of pHi recovery ( dpHi/dt ) was calculated during recovery from intracellular acidosis; A research found that activation of ERK reduced ischemia- reperfusion induced cell apoptosis, so the expression of phosphorylated ERK protein was determined by western blot.Results:1. The use of Na+/H+ exchanger inhibitor EIPA (5μM , 10μM) on the sodium-free and sodium-recovery phase, as compared with the control group, inhibited the myocardial Na+/H+ exchanger activity by 102.9% and 109.7% (P<0.05); While simvastatin in different concentrations (10 nM, 100 nM, 1000 nM) inhibited the activation of Na+/H+ exchanger was not significant, namely, -0.54%, 0.16%, 0.58% (P>0.05). Pre-incubation of simvastatin (1000 nM) for 10 min, 20 min or 30 min on the inhibition of Na+/H+ exchanger was not significant, namely, 0.28%, 0.39%, 0.31% (P>0.05).2. Using ammonium chlorid (25 mM ) induced intracellular acidosis, at the sodium-free and sodium-recovery phase, adding EIPA (5μM) and simvastatin (100 nM) to the cardiomyocytes for 3 min, total protein was extracted to detect the expression of phosphorylated ERK, total ERK as a reference . Compared with control group, sodium-free plus simvastatin group, sodium-recovery plus simvastatin group and the EIPA group, respectively, the level of ERK protein were increased by 186.9%, 137.2%, 191.3% (p<0.05). Compared with sodium-recovery plus simvastatin group , phosphorylated ERK protein level of sodium-free plus simvastatin group was increased by 29.3% (p<0.05).3. Using ammonium chlorid (25 mM) induce intracellular acidosis,at the sodium-free and sodium-recovery phase ,adding EIPA (5μM) to the cells for 3 min, total protein was extracted to detect the expression of phosphorylated ERK, total ERK as a reference. And levels of phosphorylated ERK expression in every step of the acidosis model were detected. Compared with control group, sodium-free group, sodium-recovery group and the EIPA group , respectively, levels of phosphorylated ERK protein were increased by 171.4%, 118.2%, 174.7% (p<0.05). Compared with sodium-recovery group, phosphorylated ERK protein level of sodium-free group was increased by 24.4% (p <0.05).Conclusions:1. Effects of simvastatin on Na+/H+ exchanger inhibition were not significant in cardiomyocytes;2. Intracellular acidosis stimulated the activation of phosphorylated ERK protein, and the expression of phosphorylated ERK protein levels were different from sodium-free group to sodium-recovery group. Therefore, we guessed that the concentration of extracellular sodium may affect the expression of phosphorylated ERK protein.Part twoEffects of extracellular sodium concentration on extracellular regulated protein kinaseObjective:Based on the results from the first part, we can see that the expression of ERK protein between sodium-free group and sodium-recovery group was different, we assumed that the extracellular sodium concentration may be linked to expression of phosphorylated ERK protein. We then studied the the effects of extracellular sodium concentration on extracellular regulated protein kinase.Methods:The study was performed with primary cultured neonatal rat cardiomyocytes. Intracellular pH was measured using microplate reader of multi-wavelength measurement system; Expression of phosphorylated ERK protein was determined by western blot.Results:1. Using different concentrations of extracellular sodium (135 mM, 100 mM, 50 mM, 25 mM, 0 mM) directly incubated cardiomyocytes for 10 min, total protein was extracted to detect the expression of phosphorylated ERK, total ERK as a reference .Compared with the control group, phosphorylated ERK protein levels were increased by 1.84%, 6.65%, 8.19%, 5.56%, 8.86% (p> 0.05).2. After acidification, using different concentrations of extracellular sodium (135 mM, 100 mM, 50 mM, 25 mM, 0 mM) to the cardiomyocytes for 3 min, total protein was extracted, total ERK as a reference, Compared with the 135 mM group, 100 mM group, 50 mM group, 25 mM group, 0 mM group, respectively, increased the phosphorylated ERK protein levels by 13.84%, 18.6%, 17.27% (p>0.05) and 42.27% (p<0.05).Conclusion:Effects of extracellular sodium concentration on extracellular regulated protein kinase expression were not significant.Part threeEffects of pH value on extracellular regulated protein kinase Objective: Based on the results from the second part, we can see that the effect of extracellular sodium concentration on the expreession of extracellular regulated protein kinase was not significant. We found that the pH value of 0 mM group after acidosis was about 6.67, but the 25 mM group was nearly 7.03, so we assumed that intracellular pH value may be linked to expression of phosphorylated ERK protein. We then studied the effect of intracellular pH value on extracellular regulated protein kinase. Methods:The study was performed with primary cultured neonatal rat cardiomyocytes. Intracellular pH was measured using microplate reader of multi-wavelength measurement system; Expression of phosphorylated ERK protein was determined by western blot.Results:1. Cardiomyocytes were incubated with solution of different pH values (7.2, 7.0, 6.8, 6.6) for 8 min, when the intracellular pH were the same as the extracellular solution, total protein was extracted to detect the expression of phosphorylated ERK , total ERK as a reference, Compared with the 7.2 group, phosphorylated ERK protein levels were increased by 5.22%, 7.65% (p>0.05), and 33.9% (p<0.05).2. At the terminal stage of incubation with Krebs'solution, the selective ERK inhibitor U0126 (3μM) was incubated for 5 min and was present throughout of the whole process, total protein was extracted to detect the expression of phosphorylated ERK, total ERK as a reference, Compared with the acidosis model group, phosphorylated ERK protein level was decreased by 66. 48% (p<0.05).3. At the terminal stage of incubation with Krebs'solution,the selective ERK inhibitor U0126 (3μM) was incubated for 5 min and was present throughout of the whole process. Compared with the acidosis model group, U0126 group decreased the activity of Na+/H+ exchanger by 49.46% (p<0.05).Conclusion:The phosphorylated ERK protein was senstive to the pH value around 6.6 , as in our research range (6.6~7.2) in cardiomyocytes.