PAD inhibition downregulates the cellular fibrotic behavior of senescent myofibroblasts derived from dilated cardiomyopathy

Abstract

Background: Dilated cardiomyopathy (DCM) is characterized by enlarged, weakened heart ventricles due to chronic fibrosis. Dysfunctional senescent myofibroblasts and excessive citrullination have been implicated in fibrotic diseases. Peptidylarginine deiminases (PADs) are involved in the citrullination of ECM proteins. However, their role in regulating the cellular functions of cardiac myofibroblasts in DCM, is not well understood. This study aimed to evaluate the role of PADs in the cellular biology and fibrotic behavior of myofibroblasts in DCM. Results: Aged cardiac myofibroblasts derived from dilated cardiomyopathy (DCM, N=5) and healthy (HCF, N=3) participants (35–60 years), were cultured in TGFB-conditioned medium and treated with an irreversible pan-PAD inhibitor BB-Cl-amidine. Our findings showed that, compared with HCFs, DCM myofibroblasts showed high expression of PAD-2, PAD-3, citrullinated proteins and ECM proteins (vimentin, fibronectin, actin, and b-Tubulin). BB-Cl-amidine-mediated PAD inhibition directly affected the cell biology of DCM myofibroblasts, as shown by the reduced migration and invasion of DCM myofibroblasts. It also augmented the apoptosis by activating caspase-3 and decreased senescence by regulating p-53. PAD inhibition did not affect the citrullination of vimentin or fibronectin; however, it decreased collagen 1 A expression. Conclusions: This study revealed that elevated PAD expression facilitates cellular processes mainly senescence, migration, and invasion. PAD inhibition resulted in the downregulation of these cellular functions, thereby reducing the fibrotic behavior of DCM myofibroblasts.

Keywords

• Dilated cardiomyopathy
• Fibrosis
• DCM myofibroblasts
• Peptidylarginine deiminases
• BB-Cl-amidine
• Senescence