Tousif, S. | bioRxiv Science

bioRxiv2021

Cardiac fibroblast GSK-3α mediates adverse myocardial fibrosis via IL-11 and ERK pathway

BackgroundHeart failure is the leading cause of mortality, morbidity, and healthcare expenditures worldwide. Numerous studies have implicated Glycogen Synthase Kinase-3 (GSK-3) as a promising therapeutic target for cardiovascular diseases. GSK-3 isoforms appear to play overlapping, unique, and even opposing functions in the heart. Recently our group has identified cardiac fibroblast (CF) GSK-3{beta} as a negative regulator of fibrotic remodeling in the ischemic heart. However, the role of CF-GSK-3 in myocardial fibrosis is unknown. Methods and ResultsHerein, we employed two entirely novel conditional fibroblast-specific and tamoxifen-inducible mouse models to define the role of CF-GSK-3 in fibroblast activation and myocardial fibrosis. Specifically, GSK-3 was deleted from cardiac fibroblasts or myofibroblasts with tamoxifen-inducible Tcf21- or periostin-promoter-driven Cre recombinase. At 2 months of age, WT and KO mice were subjected to cardiac injury, and heart functions were monitored by serial echocardiography. Histological analysis and morphometric studies were performed at 8 weeks post-injury. In both settings, GSK-3 deletion restricted fibrotic remodeling and improved cardiac function. To investigate underlying mechanisms, we examined the effect of GSK-3 deletion on myofibroblast transformation and pro-fibrotic TGF{beta}1-SMAD3 signaling in vitro. A significant reduction in cell migration, collagen gel contraction, and -SMA expression in TGF{beta}1 treated GSK-3 KO MEFs confirmed that GSK-3 is required for myofibroblast transformation. Surprisingly, GSK-3 deletion did not affect SMAD3 activation, indicating the pro-fibrotic role of GSK-3 is SMAD3 independent. To further delineate the underlying mechanism, total proteins were isolated from CFs of WT and KO animals at 4 weeks post-injury, and kinome profiling was performed by utilizing PamStation(R)12 high throughput microarray platform. The kinome analysis identified the downregulation of RAF family kinase activity in GSK3-KO-CFs. Moreover, mapping of significantly altered kinases against literature annotated interactions generated ERK-centric networks. Importantly, flow cytometric analysis of CFs confirmed a significant decrease in pERK levels in KO mice. Additionally, our in vitro studies demonstrated that GSK-3 deletion prevented TGF{beta}1 induced ERK activation thereby validating our findings from kinome analysis. Interestingly, IL-11, a fibroblast specific downstream effector of TGF{beta}1, was very low in GSK-3 KO MEFs as compared to WT and ERK inhibition further reduced IL-11 expression in them. All these results indicate that GSK-3 mediates pro-fibrotic response in the injured heart through IL-11 and ERK pathway. ConclusionCF-GSK-3 plays a causal role in myocardial fibrosis that could be therapeutically targeted for future clinical applications.

molecular biology↗

bioRxiv2020

Repurposing Nintedanib for Pathological Cardiac Remodeling and Dysfunction

BackgroundHeart Failure (HF) is the leading cause of death worldwide. Myocardial fibrosis, one of the clinical manifestations implicated in almost every form of heart disease, contributes significantly to HF development. However, there is no approved drug specifically designed to target cardiac fibrosis. Nintedanib (NTB) is an FDA approved tyrosine kinase inhibitor for idiopathic pulmonary fibrosis (IPF) and chronic fibrosing interstitial lung diseases (ILD). The favorable clinical outcome of NTB in IPF patients is well established. Furthermore, NTB is well tolerated in IPF patients irrespective of cardiovascular comorbidities. However, there is a lack of direct evidence to support the therapeutic efficacy and safety of NTB in cardiac diseases. Methods and ResultsWe examined the effects of NTB treatment on cardiac fibrosis and dysfunction using a murine model of HF. Specifically, 10 weeks old C57BL/6J male mice were subjected to Transverse Aortic Constriction (TAC) surgery. NTB was administered once daily by oral gavage (50mg/kg) till 16 weeks post-TAC. Cardiac function was monitored by serial echocardiography. Histological analysis and morphometric studies were performed at 16 weeks post-TAC. In the control group, systolic dysfunction started developing from 4 weeks post-surgery and progressed till 16 weeks. However, NTB treatment prevented TAC-induced cardiac functional decline. In another experiment, NTB treatment was stopped at 8 weeks, and animals were followed till 16 weeks post-TAC. Surprisingly, NTBs beneficial effect on cardiac function was maintained even after treatment interruption. NTB treatment remarkably reduced cardiac fibrosis as confirmed by Massons trichome staining and decreased expression of collagen genes (COL1A1, COL3A1). Compared to TAC group, NTB treated mice showed lower HW/TL ratio and cardiomyocyte cross-sectional area. Our in vitro studies demonstrated that NTB prevents myofibroblast transformation, TGF{beta}1-induced SMAD3 phosphorylation, and production of fibrogenic proteins (Fibronectin-1). However, NTB significantly altered vital signaling pathways in both, isolated fibroblast and cardiomyocytes, suggesting that its biological effect and underlying cardiac protection mechanisms are not limited to fibroblast and fibrosis alone. ConclusionOur findings provide a proof of concept for repurposing NTB to combat adverse myocardial fibrosis and encourage the need for further validation in large animal models and subsequent clinical development for HF patients.

pathology↗