Tanshinone I alleviates post-ischemic myocardial injury by targeting TGFBR1 and modulating the TGF-β signaling pathway

Abstract

Background: Tanshinone I (Tan I) is an essential active ingredient of the traditional cardiovascular medicine Salvia miltiorrhiza Bunge (S. miltiorrhiza). Although the protection of Tan I on cardiomyocyte has been reported, its anti-myocardial ischemia effects and mechanisms remain unknown.

Purpose: Systematic evaluation of the role of Tan I in reducing myocardial ischemia (MI) injury and elucidation of the underlying molecular mechanisms by which Tan I improves myocardial fibrosis and ventricular function in mouse MI models.

Methods: In vivo and in vitro MI models were constructed to substantiate the anti-MI effects of Tan I. Through target fishing, molecular docking, and network pharmacology investigation, the effect mechanisms and potential target proteins of Tan I against MI were predicted further. Tandem mass tags (TMT)-based quantitative proteomics, transforming growth factor beta receptor I (TGFBR1)-overexpressing lentiviral vectors, molecular dynamics (MD) simulations, biolayer interferometry (BLI), cellular thermal shift assay (CETSA), TGFBR1 kinase activity, and drug affinity responsive target stability (DARTS) assay were subsequently used to validate the anti-MI-effect mechanisms and targets of Tan I.

Results: Tan I can markedly increase the survival of oxidative stress cell models, improve intracellular environment, and inhibit the release of intracellular reactive oxygen species. Moreover, it can restore abnormal electrocardiograms, decrease myocardial infarction area, inhibit cardiac fibrosis, and reduce serum levels of key cardiac injury biomarkers in the MI mouse model. Mechanistically, Tan I considerably inhibited the phosphorylation modification levels of TGFBR1 and Smad2 and the aberrant expressions of Collagen I/III, α-smooth muscle actin, Bcl-2, and Bax proteins in MI mice. These findings were further verified in NIH-3T3 cells overexpressing TGFBR1 or activated by TGF-β1. MD simulations, CETSA, and DARTS showed that TGFBR1 binding to Tan I was relatively stable. In addition, BLI indicated that the equilibrium dissociation constant of Tan I binding TGFBR1 was 1.5 × 10^-6 M. Based on the kinase activity assay, Tan I restrained TGFBR1 with a half-maximal inhibitory concentration of 739.6 nM.

Conclusion: This work reveals for the first time that Tan I can reduce MI injury and fibrosis by modulating the TGF-β signaling pathway via targeting of TGFBR1.

Keywords: Myocardial fibrosis; Myocardial ischemia; TGF-β signaling pathway; TGFBR1; Tanshinone I.