The Differential Stereoselectivity Effects Of The Optical Enantiomers Of MN9202 And The Effects And Mechanisms Of Them On Myocardium Injury Induced By LPS

Aim In this study, we attempt to set up a resolution approach to get single enantiomer of MN9202 on a chiral stationary phase column; then, to determine the stereoselectivity effects of the optical enantiomers of MN9202 on cell shortening and relengthening, calcium transient and L-type calcium channels in rat ventricular myocytes; in addition, to pursue the effects and mechanisms of the optical enantiomers of MN9202 on myocardium injury induced by LPS. From above, we want to get a scientific evaluation for MN9202, which will be helpful for enlarging our understanding of clinic applications of dihydropyridine-type drugs.Methods①The enantiomers of MN9202 were separated on Daicel OJ-H column by optimizing conditions of chromatogram, such as the proportion of hexane or ethanol in the mobile phase.②Mechanical contraction properties and calcium transient were recorded in myocytes and assessed by video-edge detection system in absence or presence of racemic MN9202, S-(-)-MN9202 and R-(+)-MN9202.③To investigate the stereoselectivity of the optical enantiomers of MN9202 to ion channels, the actions (including ICa,L, steady-state activation and steady-state inactivation curves) of the optical enantiomers of MN9202 and racemic MN9202 were studied on L-type calciun channels in single isolateded adult rat ventricular myocytes, using the whole-cell configuration of the patch clamp technique.④Sepsis model was set up by LPS (5 mg/kg, i.v.) in anesthetized rats. The right carotid artery was connected to a pressure transducer (RM-6200) for the measurement of mean arterial blood pressure (MAP) and heart rate (HR). After recorded baseline hemodynamic parameters, animals received vehicle or LPS were monitored for 240 min. 0.5 ml blood was taken to measure the plasma levels of TNF-αby ELISA. Finally, myocardial samples were taken to stain with hematoxylin and eosin.⑤The primary cultured neonatal rat cardiomyocytes were treated with LPS, alone or in the presence of various concentrations of racemic MN9202, MN9202 enantiomers or amlodipine. The TNF-αrelease in the culture medium was measured by ELISA. TNF-αmRNA and proteins in myocytes were measured by RT-PCR and immunofluorescent staining analysis, respectively.⑥The iNOS and COX-2 mRNA or protein levels in LPS-treated cardiomyocytes at different times or cotreated with DHPs were assessed by RT-PCR and western blot assays, respectively. Regulation of phospho-Akt (pAkt) and Akt was assessed by western blot assays.Results①The chromatographic separations were performed using a OJ-H column and using a mobile phase, hexane: ethanol: triethylamine (93︰7︰0.5, V ︰V︰V). The flow-rate of the mobile phase was 0.5 ml/min, and the samples were monitored with UV detection at 237 nm. Under the optimized conditions, the enantiomers of MN9202 were well separated from the baseline and several milligrams two enantiomers, S-(-)-MN9202 and R-(+)-MN9202 were obtained.②The exposure of racemic MN9202 and S-(-)-MN9202 (1×10-8 mol/l ~ 1×10-5 mol/l) caused concentration-dependent inhibition of PTA and ?FFI in single rat ventricular myocytes. The extent of maximal percentage inhibition of PTA %baseline was significantly reduced by 73.8 % and 92.7 %, respectively. ?FFI was reduced from 0.45±0.04 to 0.07±0.02 and from 0.45±0.04 to 0.05±0.01 in presence of 1×10-5 mol/l racemic MN9202 and S-(-)-MN9202, respectively. However, R-(+)-MN9202 induced concentration-dependent increases of PTA and ?FFI in single rat ventricular myocytes. The extent of maximal percentage inhibition of PTA %baseline was significantly increased by 18.2 %, and ?FFI was increased from 0.45±0.01 to 0.58±0.05 in presence of 1×10-5 mol/l R-(+)-MN9202 (P<0.01, compared with control group).③In response to depolarizations positive to 0 mV, ICa,L density was reduced by 6.7 %,15.0 % (P<0.01), 27.5 % (P<0.01), 50.7 % (P<0.01) and 69.5 % (P<0.01), respectively in 0.03, 0.1, 0.3, 1 and 3μmol/l racemic MN9202 groups. S-(-)-MN9202 (10-6 mol/l) reduced the ICa,L density from -9.8±1.0 to -3.2±0.9 (P<0.01, 67% decrease) in response to depolarizations positive to 0 mV, exhibiting more effective antagonist activity than racemic MN9202. However, R-(+)-MN9202 (10-6 mol/l) increased the ICa,L density from -9.3±0.7 to -10.3±0.6 (P<0.01) in response to depolarizations positive to 0 mV. In ventricular myocytes, racemic MN9202, S-(-)-MN9202 and R-(+)-MN9202 didn't affect the steady-state activation curve of ICa, L. However, a shift of the midpoint of the steady-state inactivation curve was produced in the hyperpolarizing direction from ?28.8±0.2 mV to ?35.4±0.7 mV in the presence of 1μmol/l racemic MN9202 and from?28.8±0.2 mV to ?37.7±0.6 mV in the presence of 1μmol/l S-(-)-MN9202. But, in contrast, R-(+)-MN9202 at the same concentration did cause only a small shift of the midpoint of the steady-state inactivation curve (to the right) in the depolarizing direction from ?28.8±0.2 mV to ?27.1±0.6.④Administration of LPS caused a rapid fall in MAP from 119±4 to 85±2 mmHg (P<0.05)within 30 min. After 60 min, there was a continuous further fall in MAP. 10 and 30μg/kg MN9202 prevented the delayed fall in MAP observed in LPS groups. Administration of LPS resulted in an increase of heart rate, and in the MN9202 group, there was no significant difference of heart rate compared with LPS group. The injection of LPS resulted in bell-shape changes in the plasma levels of TNF-αwhich reached a peak at 60 min after LPS injection and subsequently decreased slowly (P<0.05). Treatment of 10μg/kg and 30μg/kg MN9202 decreased the TNF-αlevel in plasma. In LPS group, increase and oedema of interstitial cell, vacuolar degeneration of cardiomyocytes, inflammatory cell infiltrate have been significantly found, compared with control group. Administration of 10μg/kg and 30μg/kg MN9202 mitigated these above symptoms.⑤Neonatal rat cardiomyocytes did not release detectable amounts of TNF-αin the absence of LPS. However, in the presence of LPS, TNF-αrelease in the culture medium increased in a concentration-depended manner and mRNA levels also increased rapidly, reaching a peak at 12 h. Amlodipine, racemic MN9202 and R-(+)-MN9202, in concentrations ranging from 0. 1 to 10μmol/l, could concentration-dependently inhibit the release and mRNA expression of TNF-αin LPS-stimulated cardiomyocytes, but S-(-)-MN9202 had no obvious effect on the TNF-αrelease and mRNA expression. The inhibition rates of TNF-αrelease from LPS-stimulated neonatal rat cardiomyocytes in 1.0μmol/l amlodipine, racemic MN9202, R-(+)-MN9202 and S-(-)-MN9202 groups were 52.41, 31.62, 49.81 and 2.25 %, respectively.⑥Racemic MN9202 significantly attenuated the iNOS and TNF-αmRNA and protein levels in cardiomyocytes stimulated with LPS in a concentration-dependent manner. While the inhibition rate of 1.0μmol/l MN9202 on COX-2 expression was almost as same as the one of amlodipine, MN9202 showed more effective regulation on the iNOS levels in same concentration. Furthermore, R-MN9202 significantly depressed the levels of iNOS and COX-2, but S-MN9202 could not significantly attenuate these inflammation-relevant genes expression.⑦Compared with LPS-stimulated cells, when cardiomyocytes were cotreated with LPS and racemic MN9202, R-(+)-MN9202 or amlodipine, the phosphorylation levels of Akt were markedly increased (P<0.05), but S-(-)-MN9202 had no effect on the pAkt levels. Pre-treatment with selective inhibitors of the PI3K/Akt pathway, wortmannin or LY294002, in LPS and DHPs (racemic MN9202, R-(+)-MN9202 and amlodipine)-treated cells, Akt phosphorylation was partially inhibited. To elucidate the contribution of PI3K/Akt pathway to the regulation of TNF-α, Cox-2 and iNOS protein levels, cardiomyocytes were pretreated with wortmannin or LY294002 for 2 h and then cotreated with LPS and DHPs. The inhibition of PI3K with wortmannin or LY294002 partly attenuated the depression effects of DHPs on TNF-αprotein expression, but there was no significiant difference on the Cox-2 and iNOS expression between the groups which pre-treated with the PI3K/Akt inhibitors or not.Conclusion①The proposed chromatographic method is easy and accurate. It is reliable in the separation, preparation and quantification of MN9202.②S-(-)-MN9202 and R-(+)-MN9202 produce differential stereoselectivity effects in mechanical contraction properties (shortening and relengthening of individual myocytes), myocyte Ca2+ transients and L-type calcium channels. S-(-)-MN9202 displayed an antagonist activation, whereas R-(+)-MN9202 exerted an agonist effect.③MN9202 protected the impaired myocardium induced by LPS through inhibiting the LPS-induced expression of TNF-α, iNOS and COX-2. R-(+)-MN9202 has more markedly inhibitory effects on these LPS-induced inflammation-relevant genes expression.④The anti-TNF-αeffects of MN9202 were partly mediated by activation of Akt, downstream of the PI3K signal cascade, but anti-iNOS and COX-2 effects might be mediated by other signal pathway.