Single-cell analysis reveals lysyl oxidase (Lox)+ fibroblast subset involved in cardiac fibrosis of diabetic mice

Heyangzi Li; Xiaoqing Zhu; Xi Cao; Yicheng Lu; Jianwei Zhou; Xiaoming Zhang

doi:10.1016/j.jare.2023.01.018

Single-cell analysis reveals lysyl oxidase (Lox)⁺ fibroblast subset involved in cardiac fibrosis of diabetic mice

J Adv Res. 2023 Dec:54:223-237. doi: 10.1016/j.jare.2023.01.018. Epub 2023 Jan 24.

Authors

Heyangzi Li¹, Xiaoqing Zhu², Xi Cao¹, Yicheng Lu¹, Jianwei Zhou³, Xiaoming Zhang⁴

Affiliations

¹ Department of Basic Medicine Sciences, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310058, China.
² Department of Gynecology, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310058, China.
³ Department of Gynecology, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310058, China. Electronic address: 2195045@zju.edu.cn.
⁴ Department of Basic Medicine Sciences, and Department of Gynecology, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310058, China. Electronic address: zxm@zju.edu.cn.

Abstract

Introduction: Myocardial fibrosis and cardiac dysfunction are the main characteristics of diabetic heart disease. However, the molecular mechanisms underlying diabetic myocardial fibrosis remain unclear.

Objectives: This study aimed to investigate the heterogeneity of cardiac fibroblasts in diabetic mice and its possible mechanism in the development of diabetic myocardial fibrosis.

Methods: We established a diabetic mouse model by injecting mice with streptozotocin. The overall cell profiles in diabetic hearts were analyzed using single-cell RNA transcriptomic techniques. Cardiac function was evaluated by echocardiography. Cardiac fibrosis was assessed by Masson's trichrome and Sirius red staining. Protein expression was analyzed using Western blotting and immunofluorescence staining.

Results: A total of 11,585 cells were captured in control (Ctrl) and diabetic (DM) hearts. Twelve cell types were identified in this study. The number of fibroblasts was significantly higher in the DM hearts than in the Ctrl group. The fibroblasts were further re-clustered into nine subsets. Interestingly, cluster 4 fibroblasts were significantly increased in diabetic hearts compared with other fibroblast clusters. Lysyl oxidase (Lox) was highly expressed in DM fibroblasts (especially in cluster 4). Beta-aminopropionitrile, a Lox inhibitor, inhibited collagen expression and alleviated cardiac dysfunction in the diabetic group. Lysyl oxidase inhibition also reduced high glucose-induced collagen protein upregulation in primary fibroblasts. Moreover, a TGF-β receptor inhibitor not only prevented an increase in Lox and Col I but also inhibited the phosphorylation of Smad2/3 in fibroblasts.

Conclusions: This study revealed the heterogeneity of cardiac fibroblasts in diabetic mice for the first time. Fibroblasts with high expression of Lox (cluster 4 fibroblasts) were identified to play a crucial role in fibrosis in diabetic heart disease. The findings of this study may provide a possible therapeutic target for interstitial fibrosis.

Keywords: Single-cell RNA sequencing; diabetic cardiomyopathy; fibroblasts; lysyl oxidase.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cardiomyopathies* / metabolism
Cardiomyopathies* / pathology
Collagen / metabolism
Diabetes Mellitus, Experimental* / chemically induced
Diabetes Mellitus, Experimental* / drug therapy
Diabetes Mellitus, Experimental* / metabolism
Fibroblasts / metabolism
Fibrosis
Mice
Protein-Lysine 6-Oxidase / genetics
Protein-Lysine 6-Oxidase / metabolism
Single-Cell Analysis

Substances

Protein-Lysine 6-Oxidase
Collagen