| Background Under normal physiological conditions, activation ofβ-adrenoreceptor(β-AR) by the sympathetic nervous system stimulate Gs proteins, and then Gsαdissociation from Gβγ, the classic Gs/adenylyl cyclase (AC)/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) cascade activate , which leads to phosphorylation of various calcium regulatory proteins within the myocyte such as L-type Ca2+ channel(LTCC),ryanodine receptor 2(RyR2),phospholamban(PLB),and regulate the expression of FK506 binding protein(FKBP12.6), sarcoplasmic reticulum Ca2+ ATPase(SERCA2a) et al. Theβ-AR/Gs/AC pathway is the most powerful physiological mechanism to accurately augment cardiac contractility, So Gsαproteins may be play an important roles in this system signaling.Heart failure is a state characterized by enhanced sympathetic tone, theβ-AR/Gs/AC pathway exhibits marked alterations: down regulation or desensitization inβ-AR, and an impairment of the coupling ofβ-AR to Gs, the content and (or) activation of Gsαhas changed too, downstream mechanisms (calcium regulatory proteins) are altered. What is role of the changes of Gsαin heart failure is still unknown, is that a main factor causing further deterioration of heart function or adaptive mechanism? Moreover, previous studies in this signaling pathway mainly focused on adult animals, whether there are different manifestations in junior animals is still unclear.So this study is to knockdown of Gsαgene by RNAi in junior rat and to investigate the effects of changes of Gsαin heart failure,, and the influence of Gsαon downstream mechanisms(calcium regulatory proteins, such as RyR2,FKBP12.6,PLB,SERCA2a ) .Part 1 Construction and identification of eukaryotic expression plasmids containing short hairpin RNA targeting to gnas geneObjective To construct eukaryotic expression plasmids containing short hairpin RNA (shRNA) that target to rat gnas gene, which encodes the a-subunit of the Gs protein (Gsα).Methods Three pairs of shRNAs that target to gnas gene were designed. After annealed, the inserts were ligated into the linearized pGenesil-1.1 plasmids. The eukaryotic expression plasmids were constructed and identified using restriction enzyme analysis and sequencing analysis. And targeting no-isogeny gene was served as a negative control.Results PGenesil-1.1 plasmids were confirmed by restriction enzyme analysis and sequencing analysis in accordance with design requiring.Conclusion We successfully constructed 3 recombinant plasmids pGenesil-1.1-shRNA targeted to gnas gene, and it's helpful for further research on the role of Gsαin heart failure by RNA interference (RNAi) technique.Part 2 Selective knockdown of Gsαin primary cultured rat myocytes by RNAi in vitroObjective To selectively knockdown the expression of Gsαprotein in rat myocytes by RNA interference (RNAi),and to select the plasmids having the best effect on inhibiting Gsα.Method Eukaryotic expression plasmids containing shRNA targeting to gnas gene were constructed, then plasmids were transfected into primary cultured rat myocytes via liposome Lipofectamine? LTX & PLUS , there are plasmids carrying a nonspecific shRNA coding sequence(HK plasmids) as the negative control group and the blank control group involved. The mRNA and protein expressions of Gsαwere analyzed by RT-PCR and Western blot respectively, and were normalized to internal control gene GAPDH.Results The expressions of mRNA and protein of Gsαof test groups were markedly decreased than that of control groups (P<0.01), and the shRNA3 has the mostly inhibitory effect. While no significant difference was found in control groups.Conclusion RNAi can selectively knockdown Gsαexpression in primary cultured myocytes , and laid a foundation for future studies on Gsαin vivo.Part 3 Effect of Gsαgene knockdown by RNAi on calcium regulatory proteins in junior rat with heart failureObjective To knockdown of Gsαgene by RNAi in junior rats and investigate the effects of Gsαgene on on calcium regulatory proteins in rats with heart failure.Methods Male Wistar rats, four weeks old, were randomly divided into 3 groups, measured by the high frequency ultrasound. (1)HF group which Gsαknockdown (n=13), a mixture test plasmids shRNA3 (diluted in sterile PBS) was injected transdiaphragmatically into the hearts, and then established the animal model of chronic heart failure by fistulation of abdominal aorta and inferior vena cave. (2) HF group (n=11), Equivalent of sterile PBS was injected into the hearts, and then established the animal model of chronic heart failure by the same approach. (3)Sham group (n=9). 8 weeks after the operation, the test rats were used to test the following procedures: measurement of cardiac dimensions and ejection fraction with echocardiogram, determination of proteins expression level of RyR2,FKBP12.6,PLB,SERCA2αin cardiac tissue with Western-blot and RT-PCR .Results The echocardiogram showed that, compared with those of Sham group, many parameters of cardiac function decreased,and the expression level of RyR2, FKBP12.6 and SERCA2αalso decreased in the two HF groups, which have significant differences(P<0.01).While the expression level of PLB had no significant difference in these 3 groups (P>0.05). Compare with HF group, the Left ventricular end-systolic volume (LVESV) decreased(P<0.05), Left ventricular fraction shortening (LVFS),Left ventricular ejection fraction(LVEF)increased (P<0.05), and Left ventricular internal dimension diastole (LVIDd ),Left ventricular internal dimension systole(LVIDs),left ventricular end-diastolic volume (LVEDV) decreased, while had no statistically significant difference(P>0.05), the expression of RyR2,FKBP12.6,SERCA2αincreased(P<0.01), and FKBP12.6/ RyR2,SERCA2α/PLB increased in HF group which Gsa knockdown.Conclusion Compare with HF group, the HF group which Gsa knockdown has improvement trends in cardiac function index and relatively high-level expression of calcium regulatory proteins. Our study suggests that the alterations of Gsαmay play an adaptive reaction to the increased sympathetic nervous system activity in heart failure. |
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