Treatment of human cardiac fibroblasts with the protein arginine deiminase inhibitor BB-Cl-amidine activates the Nrf2/HO-1 signaling pathway

Aneta Stachowicz; Alia Sadiq; Brian Walker; Niveda Sundararaman; Justyna Fert-Bober

doi:10.1016/j.biopha.2023.115443

Treatment of human cardiac fibroblasts with the protein arginine deiminase inhibitor BB-Cl-amidine activates the Nrf2/HO-1 signaling pathway

Biomed Pharmacother. 2023 Nov:167:115443. doi: 10.1016/j.biopha.2023.115443. Epub 2023 Sep 11.

Authors

Aneta Stachowicz¹, Alia Sadiq², Brian Walker², Niveda Sundararaman³, Justyna Fert-Bober⁴

Affiliations

¹ Chair of Pharmacology, Jagiellonian University Medical College, Krakow, Poland; Advanced Clinical Biosystems Research Institute, Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
² Advanced Clinical Biosystems Research Institute, Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
³ Advanced Clinical Biosystems Research Institute, Precision Biomarker Laboratories, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
⁴ Advanced Clinical Biosystems Research Institute, Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA; Advanced Clinical Biosystems Research Institute, Precision Biomarker Laboratories, Cedars-Sinai Medical Center, Los Angeles, CA, USA. Electronic address: Justyna.Fert-Bober@cshs.org.

PMID: 37703660
DOI: 10.1016/j.biopha.2023.115443

Abstract

Background: Cardiac fibrosis contributes to end-stage extracellular matrix remodeling and heart failure (HF). Cardiac fibroblasts (CFs) differentiate into myofibroblasts (myoFbs) to preserve the structural integrity of the heart; however, the molecular mechanisms regulating CF transdifferentiation remain poorly understood. Protein arginine deiminase (PAD), which converts arginine to citrulline, has been shown to play a role in myocardial infarction, fibrosis, and HF. This study aimed to investigate the role of PAD in CF differentiation to myoFbs and identify the citrullinated proteins that were associated with phenotypic changes in CFs.

Results: Gene expression analysis showed that PAD1 and PAD2 isoforms, but not PAD4 isoforms, were abundant in both CFs and myoFbs, and PAD1 was significantly upregulated in myoFbs. The pan-PAD inhibitor BB-Cl-amidine (BB-Cl) downregulated the mRNA expression of PAD1 and PAD2 as well as the protein expression of the fibrosis marker COL1A1 in CFs and myoFbs. Interestingly, a proteomic approach pointed to the activation of the Nrf2/HO-1 signaling pathway upon BB-Cl treatment in CFs and myoFbs. BB-Cl administration resulted in the upregulation of HO-1 at both the gene and protein levels in CFs and myoFbs. Importantly, the protein citrullination landscape of CFs consisting of 86 novel citrullination sites associated with focal adhesion (FN1(R1054)), inflammation (TAGLN(R12)) and DNA replication (EEF2(R767)) pathways was identified.

Conclusions: In summary, we revealed that BB-Cl treatment resulted in increased HO-1 expression via the Nrf2 pathway, which could prevent excessive tissue damage, thereby leading to substantial clinical benefits for the treatment of cardiac fibrosis.

Keywords: Cardiac fibrosis; Citrullination; Heme oxygenase 1; Posttranslational modification; Protein arginine deiminase; Proteomics.