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. 2018 Apr 13;122(8):1052-1068.
doi: 10.1161/CIRCRESAHA.117.312130. Epub 2018 Mar 13.

Endoplasmic Reticulum Protein TXNDC5 Augments Myocardial Fibrosis by Facilitating Extracellular Matrix Protein Folding and Redox-Sensitive Cardiac Fibroblast Activation

Ying-Chun Shih  1 , Chao-Ling Chen  1 , Yan Zhang  1 , Rebecca L Mellor  1 , Evelyn M Kanter  1 , Yun Fang  1 , Hua-Chi Wang  1 , Chen-Ting Hung  1 , Jing-Yi Nong  1 , Hui-Ju Chen  1 , Tzu-Han Lee  1 , Yi-Shuan Tseng  1 , Chiung-Nien Chen  1 , Chau-Chung Wu  1 , Shuei-Liong Lin  1 , Kathryn A Yamada  1 , Jeanne M Nerbonne  1 , Kai-Chien Yang  2
Affiliations

Endoplasmic Reticulum Protein TXNDC5 Augments Myocardial Fibrosis by Facilitating Extracellular Matrix Protein Folding and Redox-Sensitive Cardiac Fibroblast Activation

Ying-Chun Shih et al. Circ Res. .

Abstract

Rationale: Cardiac fibrosis plays a critical role in the pathogenesis of heart failure. Excessive accumulation of extracellular matrix (ECM) resulting from cardiac fibrosis impairs cardiac contractile function and increases arrhythmogenicity. Current treatment options for cardiac fibrosis, however, are limited, and there is a clear need to identify novel mediators of cardiac fibrosis to facilitate the development of better therapeutics. Exploiting coexpression gene network analysis on RNA sequencing data from failing human heart, we identified TXNDC5 (thioredoxin domain containing 5), a cardiac fibroblast (CF)-enriched endoplasmic reticulum protein, as a potential novel mediator of cardiac fibrosis, and we completed experiments to test this hypothesis directly.

Objective: The objective of this study was to determine the functional role of TXNDC5 in the pathogenesis of cardiac fibrosis.

Methods and results: RNA sequencing and Western blot analyses revealed that TXNDC5 mRNA and protein were highly upregulated in failing human left ventricles and in hypertrophied/failing mouse left ventricle. In addition, cardiac TXNDC5 mRNA expression levels were positively correlated with those of transcripts encoding transforming growth factor β1 and ECM proteins in vivo. TXNDC5 mRNA and protein were increased in human CF (hCF) under transforming growth factor β1 stimulation in vitro. Knockdown of TXNDC5 attenuated transforming growth factor β1-induced hCF activation and ECM protein upregulation independent of SMAD3 (SMAD family member 3), whereas increasing expression of TXNDC5 triggered hCF activation and proliferation and increased ECM protein production. Further experiments showed that TXNDC5, a protein disulfide isomerase, facilitated ECM protein folding and that depletion of TXNDC5 led to ECM protein misfolding and degradation in CF. In addition, TXNDC5 promotes hCF activation and proliferation by enhancing c-Jun N-terminal kinase activity via increased reactive oxygen species, derived from NAD(P)H oxidase 4. Transforming growth factor β1-induced TXNDC5 upregulation in hCF was dependent on endoplasmic reticulum stress and activating transcription factor 6-mediated transcriptional control. Targeted disruption of Txndc5 in mice (Txndc5-/-) revealed protective effects against isoproterenol-induced cardiac hypertrophy, reduced fibrosis (by ≈70%), and markedly improved left ventricle function; post-isoproterenol left ventricular ejection fraction was 59.1±1.5 versus 40.1±2.5 (P<0.001) in Txndc5-/- versus wild-type mice, respectively.

Conclusions: The endoplasmic reticulum protein TXNDC5 promotes cardiac fibrosis by facilitating ECM protein folding and CF activation via redox-sensitive c-Jun N-terminal kinase signaling. Loss of TXNDC5 protects against β agonist-induced cardiac fibrosis and contractile dysfunction. Targeting TXNDC5, therefore, could be a powerful new therapeutic approach to mitigate excessive cardiac fibrosis, thereby improving cardiac function and outcomes in patients with heart failure.

Keywords: endoplasmic reticulum; fibrosis; heart failure; oxidative stress; sequence analysis, RNA.

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Figures

Figure 1
Figure 1. RNASeq and co-expression gene network analyses identified TXNDC5 as a potential novel mediator of cardiac fibrosis
(A) RNASeq analysis revealed upregulation of fibrogenic genes (including ECM protein genes COL1A1, COL1A2, COL3A1, ELN and FN1, matricellular protein CCN2 and genes involved in TGFβ signaling such as TGFB1, TGFB2, SMAD2 and SMAD7) and HF markers (ANF/NPPA and BNP/NPPB) in human HF, compared to NF, LV. (B) Cluster dendrogram and network heat map plot of the 15 gene modules identified by Weighted Gene Co-expression Network Analysis (WGCNA) on the human LV RNASeq data. (C) Gene ontology analysis revealed that module Turquoise was enriched in genes that are involved in the pathogenesis of cardiac fibrosis. (D) TXNDC5 expression in human LV showed strong positive correlations with TGFβ1 and ECM genes including COL1A1, ELN and FN1. The transcript (E) and protein (F) expression levels of TXNDC5 were significantly upregulated in human HF, compared to NF, LV samples (‡P<0.05, #P<0.01, *P<0.001).
Figure 2
Figure 2. TXNDC5 is highly enriched in cardiac fibroblasts and upregulated in response to TGFβ1 stimulation Transcript
(A) and protein (B) expression analyses in isolated human cardiac fibroblasts (hCF) and cardiomyocytes (hCM) revealed strong enrichment of TXNDC5 in hCF compared to hCM. (C) Immunohistochemical staining of cardiac sections from a mouse model of isoproterenol (ISO)-induced heart failure showed strong staining of TXNDC5 in the myocardial intersitium but not in myocytes (left), similar to the distribution pattern of type 1 collagen (right) (Scale bar=20 μm). TGFβ1 treatment (4 ng/ml for 48 hours) in hCF induced significant upregulation of TXNDC5 mRNA (D) and protein (E) expression, as well as of various fibrogenic proteins including COL1A1, CCN2 and ACTA2/α-SMA, compared to vehicle-treated control cells (Ctrl) (‡P<0.05, #P<0.01, *P<0.001).
Figure 3
Figure 3. Knockdown of TXNDC5 prevented TGFβ1-induced CF activation and ECM protein upregulation
(A) TGFβ1 treatment increased the proliferation rate of control hCF (transduced with shScr); knockdown of TXNDC5 (shTXNDC5) abolished TGFβ1-induced cellular proliferation in hCF. (B) Knockdown of TXNDC5 in hCF prevented TGFβ1-induced upregulation of fibrogenic proteins including αSMA, COL1A1 and CCN2 (All significant symbols indicate comparisons to baseline shScr group without TGFβ1 treatment, except the symbol above the bars, which indicates the significant level of differences between groups of TGFβ1+shScr and TGFβ1+shTXNDC5). (C) Knockdown of Txndc5 in mouse cardiac fibroblasts (mCF) also diminished TGFβ1-induced upregulation of αSMA and ECM proteins COL1A1, ELN and CCN2 (n=6 in each group, ‡P<0.05, #P<0.01, *P<0.001).
Figure 4
Figure 4. Knockdown of TXNDC5 in hCF led to accelerated ECM protein degradation owing to ECM protein misfolding and subsequent removal through ER-associated protein degradation (ERAD)
(A) A cycloheximide protein chase assay revealed accelerated degradation of COL1A1 and ELN proteins in hCF with TXNDC5 knockdown (shTXNDC5-transduced), compared to control (shScr-transduced) cells. (B) ERAD inhibitor Eey I or shVCP treatment in hCF reversed the reduction in ELN and CCN2 protein expression resulting from knockdown of TXNDC5. (C) A fluorescence resonance energy transfer (FRET)-based protein folding assay using a dual fluorescence-labeled COL1A1 construct in hCF showed significantly reduced COL1A1 FRET efficiency in cells with knockdown of TXNDC5 (shTXNDC5, n=10), compared to scrambled control (shScr, n=10), indicating reduced COL1A1 folding with TXNDC5 depletion (‡P<0.05, #P<0.01, *P<0.001).
Figure 5
Figure 5. TXNDC5-dependent activation of hCF is associated with increased JNK, but not SMAD3 or ERK, activity
(A) Overexpression of TXNDC5 in hCF was sufficient to trigger hCF activation (as reflected in increased αSMA and POSTN protein levels), and COL1A1 production. Phosphorylation of SMAD3 was not affected by ectopic TXNDC5 expression. (B) Forced expression of TXNDC5 led to significantly increased secretion of type 1 collagen (COL1A1) from hCF. (C) Knockdown of TXNDC5 in hCF abrogated TGFβ1-induced phosphorylation of JNK, but not of SMAD3 or ERK. Phosphorylated JNK, SMAD3 and ERK were expressed relative to total JNK, SMAD3 and ERK, respectively (#P<0.01, *P<0.001).
Figure 6
Figure 6. TXNDC5-mediated activation and proliferation of hCF require JNK activity triggered by NOX-derived ROS
(A) Treatment with a JNK inhibitor (SP600125, 10 μmol/L), PDI inhibitor (16F16, 20 μmol/L) or ROS scavenger N-acetylcysteine (NAC, 15 mmol/L) abolished upregulation of αSMA and POSTN (markers for hCF activation and myofibroblast formation) and COL1A1 induced by ectopic TXNDC5 expression in hCF. Note that 16F16 and NAC treatment also reduced JNK phosphorylation resulting from TXNDC5 overexpression. (B) Knockdown of TXNDC5 abolished TGFβ1-induced hCF proliferation, whereas overexpression of TXNDC5 was sufficient to induce hCF proliferation. Pharmacological inhibition of JNK (with SP600125), PDI (with 16F16) or ROS (with NAC) abrogated hCF proliferation induced by TXNDC5 overexpression. (C) Knockdown of TXNDC5 abrogated TGFβ1-induced ROS elevation in hCF, whereas TXNDC5 overexpression was sufficient to increase ROS levels, which could be abolished by the PDI inhibitor, 16F16, or the NOX inhibitor, apocynin (2 mmol/L). (D) Representative photomicrographs illustrating increased ROS levels (measured using DCFDA fluorescence signal intensity) in hCF with ectopic TXNDC5 expression, which is diminished by the NOX inhibitor apocynin and by the PDI inhibitor, 16F16 (‡P<0.05, #P<0.01, *P<0.001).
Figure 7
Figure 7. TGFβ1 induces TXNDC5 expression in CF through TGFβ1-ER stress-ATF6 signaling axis
(A) TGFβ1 treatment in hCF increased ER stress (as evidenced by the upregulated ER stress markers ATF6 p50 and p90) and TXNDC5 protein expression levels, which could be abrogated by the treatment with general ER stress inhibitors 4-phenylbutyrate (4-PBA, 0.5 mmol/L) or tauroursodeoxycholic acid (TUDCA, 0.5 mmol/L) (B) Knockdown of ATF6 in hCF prevented the upregulation of TXNDC5 mRNA in response to TGFβ1 stimulation. (C) Schematic illustration of the mouse Txndc5 promoter luciferase reporter construct, which contains an ATF6 binding site (TGACGTGG, +773∼+780). Deletion of the ATF6 binding site significantly reduced TGFβ1-induced transcriptional activity of the Txndc5 promoter in the absence or presence of TGFβ1. (D) Electrophoretic mobility shift assay showed biotin-labeled Txndc5 promoter probe containing ATF6 binding site was shifted (lane B) when treated with nuclear extract from NIH-3T3 fibroblasts with ectopic ATF6 expression. Unlabeled Txndc5 promoter DNA was used as competitor (lane C) and revealed the specificity of ATF6 binding to Txndc5 promoter. Biotin-labeled Txndc5 promoter probe with ATF6 binding site deletion failed to interact with ATF6 (lane D, E).(#P<0.01, *P<0.001).
Figure 8
Figure 8. Targeted deletion of Txndc5 protects against isoproterenol-induced cardiac hypertrophy, fibrosis and contractile dysfunction
(A) Isoproterenol (ISO, 30 mg/kg/day subcutaneously for 10 days) injection led to marked increase in HW/BW ratio, an indicator of cardiac hypertrophy, in WT, but not in Txndc5-/-, mice. (B) Knockout of Txndc5 attenuated the extent of myocardial fibrosis induced by ISO injection. (C) ISO treatment led to significantly increased fibrogenic proteins (including COL1A1, ELN, CCN2, αSMA, POSTN) and p-JNK expression in WT, but not in Txndc5-/-, mouse LV. (D) Loss of Txndc5 also significantly reduced the proliferation capacity of mCF at baseline and in response to TGFβ1 stimulation. (E) Schematic summary of the proposed profibrotic mechanisms by which TXNDC5 contributes to cardiac fibrosis.(‡P<0.05, #P<0.001, *P<0.001).
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