Yoda1 Inhibits TGFβ-Induced Cardiac Fibroblast Activation via a BRD4-Dependent Pathway

Abstract

Fibrosis represents a pivotal pathological process in numerous diseases, characterized by excessive deposition of extracellular matrix (ECM) that disrupts normal tissue architecture and function. In the heart, cardiac fibrosis significantly impairs both structural integrity and functional capacity, contributing to the progression of heart failure. Central to this process are cardiac fibroblasts (CFs), which, upon activation, differentiate into contractile myofibroblasts, driving pathological ECM accumulation. Transforming growth factor-beta (TGFβ) is a well-established regulator of fibroblast activation; however, the precise molecular mechanisms, particularly the involvement of ion channels, remain poorly understood. Emerging evidence highlights the regulatory role of ion channels, including calcium-activated potassium (K_Ca) channels, in fibroblast activation. This study elucidates the role of ion channels and investigates the mechanism by which Yoda1, an agonist of the mechanosensitive ion channel Piezo1, modulates TGFβ-induced fibroblast activation. Using NIH/3T3 fibroblasts, we demonstrated that TGFβ-induced activation is regulated by tetraethylammonium (TEA)-sensitive potassium channels, but not by specific K⁺ channel subtypes such as BK, SK, or IK channels. Intriguingly, Yoda1 was found to inhibit TGFβ-induced fibroblast activation through a Piezo1-independent mechanism. Transcriptomic analysis revealed that Yoda1 modulates fibroblast activation by altering gene expression pathways associated with fibrotic processes. Bromodomain-containing protein 4 (BRD4) was identified as a critical mediator of Yoda1's effects, as pharmacological inhibition of BRD4 with JQ1 or ZL0454 suppressed TGFβ-induced expression of the fibroblast activation marker Periostin (Postn). Conversely, BRD4 overexpression attenuated the inhibitory effects of Yoda1 in both mouse and rat CFs. These results provide novel insights into the pharmacological modulation of TGFβ-induced cardiac fibroblast activation and highlight promising therapeutic targets for the treatment of fibrosis-related cardiac pathologies.

Keywords: BRD4; Periostin; Yoda1; cardiac fibroblast; fibroblast activation; ion channels; mechanosensitive channels.

Figure 1

K⁺ channels do not significantly contribute to TGFβ-induced NIH/3T3 fibroblast activation. Bar graphs showing the expression of the activated fibroblast marker Periostin following treatment with (A) tetraethylammonium (TEA;10 mM), which inhibits both voltage-dependent and calcium-activated channels, (B) 4-AP (2 mM), which selectively inhibits voltage-dependent potassium (K_v) channels, (C) iberiotoxin (IbTX; 100 nM), which inhibits large-conductance calcium-activated potassium channels (BK), (D) apamin (100 nM), which inhibits small-conductance calcium-activated potassium channels (SK), (E) TRAM-34 (100 nM), which inhibits intermediate-conductance calcium-activated potassium channels (IK, K_Ca3.1), and (F) BaCl₂ (10 µM), which inhibits inward-rectifying potassium channels (K_ir). Results are presented as mean ± SE. Statistical significance between groups was assessed using one-way ANOVA. A p-value ≤ 0.05 was considered statistically significant; ** = p ≤ 0.01. NT = no treatment.

Figure 2

TGFβ induces a signature of fibrotic gene expression in NIH/3T3 fibroblasts, which is reversed by Yoda1 in a Piezo1-independent mechanism. (A) The volcano plot illustrates the genes that are significantly upregulated (orange) or downregulated (blue) in response to TGFβ. RNA sequencing revealed that 2110 genes were differentially expressed in TGFβ-treated NIH/3T3 fibroblasts compared to control, with 856 genes upregulated and 1254 genes downregulated. (B) Ingenuity Pathway Analysis (IPA) mapped the signature of differentially expressed genes to known signaling pathways. The five best matching pathways are displayed, where the teal blue bar indicates downregulation and the red bars represent upregulation, based on the differential gene expression in the data set. The size of the bubble corresponds to the number of overlapped genes in respective biological pathways. (C) IPA was used to correlate differentially expressed genes associated with cell functions and disease processes. The network illustrates the expression pattern of genes and their effects on associated pathways. Blue represents downregulation/inhibition, while orange denotes upregulation. (D,E) Bar graphs showing the effects of GsMTx4, a specific inhibitor of Piezo1, and Yoda1, a Piezo1 agonist, on the expression of Periostin with or without TGFβ treatment in NIH/3T3 fibroblasts. (F) Bar graph demonstrating the effect of Yoda1 alone or in combination with GsMTx4 on the TGFβ-induced Periostin expression. Results are presented as mean ± SE. Statistical significance was determined using one-way ANOVA, with p value ≤ 0.05 considered statistically significant. * = p ≤ 0.05. NT = no treatment.

Figure 3

Piezo1 knockdown does not significantly affect Yoda1-mediated inhibition of TGFβ-induced fibroblast activation: (A) Bar graph represents the siPiezo1 (200 nM)-mediated inhibition of the Piezo1 in cardiac fibroblast. (B) Bar graph demonstrates the Periostin expression in the different study groups (no treatment, TGFβ, or TGFβ + Yoda1), with or without Piezo1 knockdown. (C) Bar graph demonstrates the αSMA expression in the different study groups (no treatment, TGFβ, or TGFβ + Yoda1), with or without Piezo1 knockdown. Results are presented as mean ± SE. Statistical significance was determined using one-way ANOVA, with p value ≤ 0.05 considered statistically significant. * = p ≤ 0.05. ** = p ≤ 0.01. *** = p ≤ 0.001. **** = p < 0.0001. ns = non-significant. NT = no treatment. TY = TGFβ + Yoda1.

Figure 4

Gene expression profiling in NIH/3T3 fibroblasts: Effects of TGFβ and Yoda1. (A) The Venn diagram illustrates the overlap of upregulated and downregulated genes across the different study groups. Volcano plots comparing genes upregulated or downregulated in response to (B) Yoda1 versus control and (C) TGFβ versus TGFβ + Yoda1 in NIH/3T3 fibroblasts. The orange dots represent significantly upregulated or downregulated genes, whereas blue dots represent the genes with no significant difference in expression. (D) The heatmap displays the gene expression profiles of NIH/3T3 fibroblasts across the study groups: control, Yoda1, TGFβ, and TGFβ + Yoda1. NT = no treatment.

Figure 5

Inhibition of BRD4 had similar effects on NIH/3T3 fibroblast activation as Yoda1. (A) IPA was used to compare the top pathways affected in TGFβ + Yoda1 versus TGFβ (1) and versus normal control (synovial tissue) with JQ1 (a BRD4 inhibitor). (B) Graphical representation of the putative regulation of BRD4. Red arrows indicate upregulation, while question marks denote gaps in current knowledge. (C) The heatmap illustrates the relative expression levels of Periostin, Meox1, and BRD4 in TGFβ + Yoda1-treated cells compared to TGFβ alone. (D,E) Bar graphs showing the effects of JQ1 on the expression of Periostin and Meox1 with or without TGFβ treatment in NIH/3T3 fibroblasts. (F,G) Bar graphs showing the effects of ZL0454 on the expression of Periostin and Meox1 with or without TGFβ treatment in NIH/3T3 fibroblasts. Results are presented as mean ± SE. Statistical significance was determined using one-way ANOVA, with p value ≤ 0.05 considered statistically significant. * = p ≤ 0.05. NT = no treatment.

Figure 6

BRD4 overexpression mitigates the Yoda1-mediated inhibition of Periostin expression in cardiac fibroblasts. Bar graphs showing BRD4 expression in (A) mouse cardiac fibroblasts (CFs) and (B) rat CFs transduced with adeno-GFP or adeno-BRD4 and treated with TGFβ, Yoda1, or a combination. Bar graphs depicting Periostin expression in (C) mouse CFs and (D) rat CFs transduced with adeno-GFP or adeno-BRD4 and treated with TGFβ, Yoda1, or both. Results are presented as mean ± SE. Statistical significance was calculated using one-way ANOVA, with p ≤ 0.05 considered statistically significant. * = p ≤ 0.05. NT = no treatment; Y + T = Yoda1 + TGFβ.