Tetrahydrobiopterin Improves Left Ventricular Diastolic Function By Upregulating Protein Kinase C ε Signaling Pathway In Desoxycorticosterone Acetate-salt Hypertensive Mice

Objective To identify the influence of tetrahydrobiopterin (BH4) on left ventricular diastolic function and the expression of protein kinase Cε(PKCε) in desoxycorticosterone acetate (DOCA)-salt hypertensive mice.Method Male C57BL/6 mice were randomly divided into operation group (n=50) and control group(n=40). Mice in operation group were resected the left kidney and implanted DOCA pill under the neck skin. After operation, mice were fed with normal feed and 1.05% saline water. Six mice died during nursing and 44 others were randomly further divided into control group (n=22) as well as control+BH4 group (n=22,2.5% BH4,0.1 ml/10 g intragastrical administration for 7 d) until the 14th day. Mice in control group were only separated the left kidney instead of resecting it, and placebo pill was put in the same way. After operation, mice were fed with normal fodder and normal water, then all of them were randomly separated into SHAM group (n=20) and SHAM +BH4 group (n=20). At 21th day, all groups were experiments as follows:1. Diastolic blood pressure(DBP) and systolic blood pressure(SBP) of mice tail were measured by arterial pressure(n=10).2. The cardiac structural and functional parameters, included left ventricular early diastolic mitral annular velocity(E’), left ventricular early diastolic filling velocity(E) and the ratio of left-ventricular early diastolic mitral annular velocity to mitral annular velocity at atrial contraction(E’/A’), in hypertensive mice were measured by color doppler echocardiography and tissue doppler imaging(n=10). 3. The hemodynamic outcomes such as heart rate, Tau index, left ventricular end-diastolic pressure(LVEDP), left ventricular end systolic pressure (LVESP), end-systolic pressure-volume relation(ESPVR) and end-diastolic pressure-volume relation (EDPVR) were tested by invasive hemodynamic monitor(n=6).At 22th day after operation, all the mice were executed and cardiac tissues were experimented separately with following methods: ①Cyclic guanosine monophosphate(cGMP), malonaldehyde and superoxide dismutase(SOD) were analyzed by enzyme linked immunosorbent assay(ELASA). ② The levels of BH4 and dihydrobiopterin (BH2) were confirmed by high-performance liquid chromatography(HPLC). ③lasma angiotensin II was tested by radioimmunoassay.④α-SA, TGFβ1, P-MHC and so on were measured by real time-polymerase chain reaction. ⑤ Vascular endothelial growth factor (VEGF), phospholamban(PLB), protein kinase C zeta(PKC-zeta), B cell lymphoma/lewkmia-2(BCL-2), Bcl-2 and associated X protein(Bax) were experimented by immunohistochemical method. ⑥ Expression of endothelial nitric oxide synthase (eNOS) and PKCε were confirmed by Western-blot.Results ①As compared to SHAM group, both SBP and DBP in control group were all increased(P<0.05), but for control+BH4 group and control group, there was no statistical differences in blood pressure (BP)(P>0.05). ②The E/E’,EDPVR and Tau index were increased in control group when compared with SHAM group(all P<0.05). ③ After gavaged with 2.5% BH4, EDPVR and Tau index in control+BH4 group were reduced contrasted to control group (all P<0.05).④SOD and nitric oxide(NO) in control group were reduced when compared with SHAM group(all P<0.05), after gavaged with 2.5% BH4, SOD and NO added(all P<0.05). ⑤adioimmunoassay results showed that Angll in control group was more than that in control+BH4 group, and BH4 can reduce it when it was superfluous produced(all P<0.05).⑥When compared with SHAM group, α-SA, PKCε and β-MHC in control group were decreased, but after intervened with BH4, these genes express aggrandized(all P<0.05). ⑦Result of immunohistochemical experiment indicated that VEGF, PLB, Serca2 and Bax in control group were produced more, Phospho-PLB and BCL-2 were expressed less than SHAM group. After treated with BH4, this consequence could be improved in control+BH4 group and it also can be improved in SHAM+BH4 group(all P<0.05). ⑧The consequences of western-blot indicated that as compared to SHAM group, amount of PKCε in control group was decreased(P<0.05) and amount of PKCε in control+BH4 group was increased compared with control group(P<0.05).Conclusion BH4 had little effect on BP but could improve left ventricular diastolic dysfunction in hypertensive mice, which was related to lowering the levels of oxidative stress, increasing amounts of NO by up-regulated PKCε signaling pathway.