The Study On The Mutation Of Mitofusin 2 Trigger The Apoptosis Of VSMC

Objective To study the effect of PKA phosphorylation site's mutation of mitofusin 2(Mfn2) on apoptosis of vascular smooth muscle cells.Methods Using the Adv-Mfn2, Adv-Mfn2-S442A,Adv-Mfn2-S442D and Adv-LacZ infected the vascular smooth muscle cells (VSMC) of rat, and 60 hours later, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL) staining was used to detect the apoptosis cells. Rat model of carotid artery balloon injury was established and infected with either Adv-LacZ, Adv-Mfn2, Adv-Mfn2-S442A or Adv-Mfn2-S442D from the periarterial sheathes of vessels, while injected PBS as uninfected group and sham operation as normal control group. To sacrifice the rat at day 5 and 14 after balloon injury, immunohistochemistry staining was performed to measure the expression of Mfn2.TUNEL staining was used to detect the apoptosis cells and HE staining was used to observe the change of vascular morphology.Results Sixty hours after infection, the apoptosis cells of Adv-Mfn2, Adv-Mfn2-S442A groups were markly increased. 5 days after surgery, the Mfn2 protein level was significantly increased in arteries infected with Adv-Mfn2, Adv-Mfn2-S442A and Adv-Mfn2-S442D compared with those from other groups. The percentage of TUNEL- positive cells in Adv-Mfn2, Adv-Mfn2-S442A groups were markedly increased compared with those in groups of Control, Adv-LacZ and Adv-Mfn2-S442D groups(P<0.01). Compared with the Adv-Mfn2 group, the TUNEL- positive cells of Adv-Mfn2-S442A group was increased more (P<0.01). 14 days after surgery, the ratio of intimal area to media area(I/M) was markedly attenuated in Adv-Mfn2 and Adv-Mfn2-S442A groups compared with other groups(P<0.01). And compared with the Adv-Mfn2 group, the I/M of Adv-Mfn2-S442A group was reduced more (P<0.01).Conclusion Mfn2-S442A has stronger promoting effect on the apoptosis of VSMCs than Mfn2,while Mfn2-S442D has no effect.The Mfn2 has more then one PKA phosphorylation site, and this PKA phosphorylation site may be influence the fuction of Mfn2 by changing the protein struction.