The effects of cortisol and TGF-b1 on glucocorticoid receptor expression in Human Cardiac Fibroblasts

Abstract

Introduction: Cardiovascular disorders such as heart failure, account for a significant proportion of morbidity and mortality across the globe. The mechanism driving heart failure is complex and can arise from several different pathological events, including a myocardial infarction; ultimately resulting in excessive fibrosis of the heart and a substantial reduction in cardiac compliance. Elevation in inflammatory mediators such as cortisol and TGF-β1 have been implicated in the progression of heart failure, and their interactions with one of the primary cell types of the heart, the cardiac fibroblast, has been linked to an increase in cardiac fibrosis. The cardiac fibroblast, while it plays a substantial role in wound repair and fibrosis, its activity is upregulated under stress when it is transformed to a myofibroblast phenotype. While TGF-β1 signalling on cardiac fibroblast and its effect on transforming cardiac fibroblasts to myofibroblasts is well defined the role of cortisol in heart failure and specifically on fibroblast function has not been studied. Nor has any interactions between the two compounds been explored in an in vitro model. Experimental Aims: This study aimed to evaluate the effects of the stress and inflammatory mediators, cortisol and TGF-β1, on cell metabolic activity and cytotoxicity measured using an MTT, CCK-8, LDH assays and an object count via plate reader. The RNA expression of fibrosis genes and protein expression of α-smooth muscle actin (α-SMA) and the glucocorticoid receptor (GR) was also measured, using NanoString nCounter analysis and western blot respectively. All of these experiments were completed using primary human cardiac fibroblasts, with the purpose of determining the interaction between stress and inflammatory mediators on cardiac fibroblast function to establish a possible contributor to the pathology of heart failure. Methods: Primary human cardiac fibroblasts were initially cultured in cardiac fibroblast specific media and subsequently grown in DMEM with 10% FBS. The MTT, CCK-8, LDH assay and object count analysis was undertaken after the cells were treated with the pharmacological treatments; hydrocortisone (1) (1μM and 0.5μM), mifepristone (5nM), TGF-β1 (5ng/ml), SB431542 (2μM) and the associated vehicle controls (ethanol [0.1% v/v])and (DMSO [0.2% v/v]) for the duration of 48 hours. For the RNA analysis using Nanostring technology, whole cell lysates were initially used after treatment of the above pharmacological compounds minus HC (0.5μM). Due to not enough RNA extraction in the first experiment, DMSO, mifepristone and TGF-β1 treatment groups were repeated using isolated total RNA using Nanostring technology. Lastly, the protein was extracted from the human cardiac fibroblasts and quantified by BCA assay before assessing for the presence of α-SMA and the GR, GRα-A and its splice variant GR-β using western blot analysis. Mouse small intestine was used a positive control for α-SMA, A549 cells were used as a positive control for GR and Glyceraldehyde 3-phosphate dehydrogenase was used as a loading control. All data was presented as ± standard error of the mean and oneway ANOVA with Tukey’s post-Hoc analysis was used to assess for statistical differences, this was achieved using GraphPad Prism Version 9.0 with a p value of 0.05 as the cut off for significance. Results: The MTT data showed groups containing HC (0.5μM) plus mifepristone, HC (1μM) plus TGF-β1, and TGF-β1 in the absence and presence of the antagonist as a significant decrease in metabolic activity (p 0.05). The CCK-8 assay was used at a 24 and 48 hour time point, and almost all treatment groups showed a significant decrease in metabolic activity (p<0.05) which matched the trends seen by the MTT data. The LDH assay demonstrated that the drug treatments caused low LDH release compared with respective controls, all treatment groups except for, SB431542 (2μM) and TGF-β1 (5ng/ml) plus SB431542 (2μM), showed a significant decrease (p<0.05). Indicating that the drug treatments while decreasing metabolic activity were not causing significant cell death. The RNA analysis using Nanostring nCounter analysis found that 48 hours of HC treatment on human cardiac fibroblasts did not cause any change in the expression of genes associated with fibrosis (p>0.05), except in collagen type I and collagen type III where a significant decrease was observed the presence of HC (1μM) plus mifepristone (p<0.05). TGF-β1 treatment of human cardiac fibroblasts caused a significant decrease in RNA expression of genes that are related to fibrotic activity (p<0.05). Following 48 hours of drug treatment on human cardiac fibroblasts the relative protein expression of α-SMA failed to reach any significance (p>0.05). With respect to the GR, protein expression for both the GRα-A and its splice variant GR-β was assessed. GRα-A protein expression significantly increased in human cardiac fibroblasts after treatment with HC (1μM and 0.5μM) and mifepristone for 48 hours (p<0.05). Likewise, treatment with TGF-β1, SB431542 and the combined treatment all resulted in a significant increase in GRα-A protein expression, (p<0.05). The GR-β protein expression failed to reach a significant increase after HC (1μM) treatment but was significantly increased after HC (1μM and 0.5μM) plus mifepristone (p<0.05). Moreover, TGF-β1, SB431542 and the combined treatment significantly increased (p<0.05) GR-β protein expression after 48 hours of treatment. Conclusion: This study investigated the independent and combined effect of HC and TGF-β1 on human cardiac fibroblasts, to assess if there were changes to RNA expression of fibrosis related genes, as well as changes to the protein expression of α-SMA and the GR. As a result of the chronic exposure to HC and TGF-β1, no significant changes were detected in the RNA or protein expression of α-SMA suggesting that these cells did not transform into myofibroblasts. Several fibrosis related genes decreased after TGF-β1 exposure, which was a novel finding in the cardiac fibroblasts. There were no substantial changes to the RNA expression of the GR, however, significant increases in the protein expression of the GRα-A and the splice variant GR-β occurred in human cardiac fibroblasts after treatment with HC and TGF-β1 for 48 hours. These results indicate that elevations in the stress and inflammatory mediators, while not influencing transformation of the cardiac fibroblasts, are having an impact on the fibrotic activity of the cell as well as its response to glucocorticoid signalling. These distinct changes seen in the human cardiac fibroblast may play a role in the progression of heart failure.