Ubiquitin C-terminal hydrolase 1 inhibits the ubiquitination of CaMK Ⅱ and promots the occurrence of atrial fibrillation in rats

Abstract

Objective To analyze the role and molecular mechanisms underlying of ubiquitin C-terminal hydrolase 1 (UCHL1) in promoting atrial fibrillation (AF). Methods Forty 8-week-old male SD rats (240~260 g) were randomly divided into four groups, with 10 rats in each group: Sham+DMSO group, Sham+ LDN (the inhibitor of UCHL1) group, AF+DMSO group and AF+LDN group. AF model was induced by subcutaneously implanted osmotic micropump of angiotensin Ⅱ (AngⅡ) in both AF+DMSO group and AF+LDN group. After 4 weeks, the duration of AF was monitored and recorded by electrocardiography. The level of fibrosis was observed by Masson-trichrome staining of left atrial tissues. Heart function was evaluated by ultrasonic cardiography. Neonatal rat cardiomyocytes (NRCM) were isolated from the hearts of SD rats aged 1~3 d of which effect was verified by overexpression or knockdown of UCHL1 in NRCM cells. Immunoprecipitation mass spectrometry was used to evaluate the functional regulatory mechanism of UCHL1 on calcium/calmodulin-dependent protein kinaseⅡ (CaMKⅡ) in NRCM. The expression of fibrotic protein was analyzed by Western blot assay. Results The expression of UCHL1 protein was increased in cardiac tissues of AF rats and in AngⅡ-treated NRCM. Compared with the AF+DMSO group, the cardiac ejection fraction (EF) and fractional shortening (FS) of the AF+LDN group were significantly increased (P < 0.01), while the degree of fibrosis, expression of α-SMA and ColⅠ and duration of AF were significantly decreased(P < 0.01). In vitro, inhibition and knockdown of UCHL1 attenuated AngⅡ-induced fibrosis of NRCM (P < 0.05). Immunoprecipitation mass spectrometry revealed that UCHL1 interacted with CaMKⅡ in NRCM simulated by AngⅡand promoted the upregulation of UCHL1 through deubiquitination. CaMKⅡ overexpression reduced the inhibitory effect of UCHL1 knockdown on the fibrotic response in AngⅡ-induced NRCM (P < 0.05). Conclusion UCHL1 interacts with CaMKⅡ and upregulates CaMKⅡ through deubiquitination, thereby promoting cardiac fibrosis in AF.

Keywords

• ubiquitin c-terminal hydrolase 1
• ca2+/calmodulin-dependent protein kinase ⅱ
• atrial fibrillation
• cardiac fibrosis