Liao Li-Zhen1, Zhi-Chong Chen2, Sui-Sui Wang1, Wen-Bin Liu1, Xiao-Dong Zhuang3. 1. Guangdong Engineering Research Center for Light and Health, Guangzhou Higher Education Mega Center, Guangzhou, Guangdong, PR China; Guangdong Provincial Key Laboratory of Pharmaceutical Bioactive Substances, Guangdong Pharmaceutical University, Guangzhou Higher Education Mega Center, Guangzhou, Guangdong, PR China. 2. Cardiovascular Department, The Sixth Affiliated Hospital of Sun Yat-sen University, No. 26, Erheng Road, Yuan Village, Tianhe District, Guangzhou, Guangdong Province, PR China. 3. Cardiology Department, The First Affiliated Hospital of Sun Yat-sen University, 58 Zhongshan 2nd Road, Guangzhou, Guangdong, PR China. Electronic address: zhuangxd3@mail.sysu.edu.cn.
Abstract
OBJECTIVE: In this study, it was hypothesized that klotho deficiency plays an essential role in cardiac ageing in vivo and demonstrated that supplementation with exogenous klotho protects against cardiomyocyte ageing in vitro. METHODS: We measured the lifespan of wild-type (WT) and klotho-hypomorphic mutant (KL-/-) mice and recorded the cardiac function of the mice through echocardiography. We used immunofluorescence staining to detect the LC3B (microtubule-associated protein light chain 3 B), Beclin 1, Bax and Bcl 2 proteins. In vitro, H9c2 cells were incubated with different levels of D-galactose (D-gal) with or without klotho. SA-β-galactosidase staining and western blotting were performed to detect ageing-associated proteins (P53, P21 and P16), autophagy-associated proteins (LC3 II/LC3 I and Beclin 1) and apoptosis-associated proteins (Bax and Bcl 2). Moreover, one-step TUNEL apoptosis, CCK-8, cell morphology, Hoechst 33258 staining, lactate dehydrogenase (LDH) release, and caspase-3 activity assays were performed, and intracellular reactive oxygen species (ROS) levels were measured. RESULTS: Genetic klotho deficiency decreased lifespan and cardiac function in mice, impaired autophagic activity and increased apoptotic activity. Exogenous klotho attenuated cardiomyocyte ageing and reversed changes in autophagic and apoptotic activity caused by D-gal. Moreover, klotho supplementation prevented D-gal-induced oxidative stress and cytotoxicity. CONCLUSIONS: Klotho might have a protective effect on cardiac ageing via autophagy activation and apoptosis inhibition.
OBJECTIVE: In this study, it was hypothesized that klotho deficiency plays an essential role in cardiac ageing in vivo and demonstrated that supplementation with exogenous klotho protects against cardiomyocyte ageing in vitro. METHODS: We measured the lifespan of wild-type (WT) and klotho-hypomorphic mutant (KL-/-) mice and recorded the cardiac function of the mice through echocardiography. We used immunofluorescence staining to detect the LC3B (microtubule-associated protein light chain 3 B), Beclin 1, Bax and Bcl 2 proteins. In vitro, H9c2 cells were incubated with different levels of D-galactose (D-gal) with or without klotho. SA-β-galactosidase staining and western blotting were performed to detect ageing-associated proteins (P53, P21 and P16), autophagy-associated proteins (LC3 II/LC3 I and Beclin 1) and apoptosis-associated proteins (Bax and Bcl 2). Moreover, one-step TUNEL apoptosis, CCK-8, cell morphology, Hoechst 33258 staining, lactate dehydrogenase (LDH) release, and caspase-3 activity assays were performed, and intracellular reactive oxygen species (ROS) levels were measured. RESULTS: Genetic klotho deficiency decreased lifespan and cardiac function in mice, impaired autophagic activity and increased apoptotic activity. Exogenous klotho attenuated cardiomyocyte ageing and reversed changes in autophagic and apoptotic activity caused by D-gal. Moreover, klotho supplementation prevented D-gal-induced oxidative stress and cytotoxicity. CONCLUSIONS: Klotho might have a protective effect on cardiac ageing via autophagy activation and apoptosis inhibition.