Single-nucleus analysis reveals Dengzhan Shengmai capsule prevents cardiac fibrosis after myocardial infarction in rat by targeting Ltbp2+ fibroblast subset

Abstract

Background: Cardiac fibrosis is a distinguishing hallmark of ischemic heart diseases with limited therapeutic options due to the incomplete comprehension of cellular heterogeneity. Our previous research demonstrated that Dengzhan Shengmai capsule (DZSMC) could significantly attenuate myocardial fibrosis, yet its cell-type-specific targets remain elusive.

Objectives: This work aimed to investigate the mechanisms of DZSMC on cardiac fibrosis in post-myocardial infarction (MI) rats at single-cell resolution.

Methods: MI rats were produced by ligating the left anterior descending artery. DZSMC (97.2 mg/kg) and captopril (4.5 mg/kg) were administered orally for four weeks following MI. Cardiac function was assessed by echocardiography. Histopathological analysis was employed via hematoxylin-eosin and Masson's trichrome staining. The overall cell profiles in MI rat hearts were analyzed by single-nucleus RNA sequencing (snRNA-seq). In vitro, the role of latent transforming growth factor beta binding protein 2 (Ltbp2) was confirmed by small interfering RNA targeting Ltbp2 (siLtbp2) in primary cardiac fibroblasts (CFs).

Results: DZSMC substantially enhanced cardiac function and attenuated pathological myocardial fibrosis in post-MI rats. snRNA-seq analysis of 28,317 nuclei from Sham, Model and DZSMC group hearts revealed ten distinct cell types. Notably, CFs exhibited the most pronounced transcriptomic alterations after DZSMC intervention. Re-clustering of CFs identified eight subsets. Interestingly, cluster 5 CFs specifically exhibited high expression of Ltbp2, along with elevated levels of Tgfb1, col1a1, and col3a1. This sub-population was functionally enriched in extracellular matrix organization and TGF-β signaling. Ltbp2 knockdown attenuated angiotensin II (Ang II) induced CFs activation and down-regulated the expression of TGF-β and pSmad3. Furthermore, DZSMC treatment significantly inhibited Ltbp2 expression and TGF-β signaling both in vitro and in vivo.

Conclusions: This study reveals Ltbp2 as a novel fibroblast-specific target for cardiac fibrosis and DZSMC exerts its anti-fibrotic effects by targeting Ltbp2⁺ CFs.

Keywords: Cardiac fibrosis; Cellular heterogeneity; Dengzhan shengmai capsule; latent transforming growth factor beta binding protein 2; snRNA-seq.