Acute Hyperglycemia Exacerbates Hemorrhagic Transformation after Embolic Stroke and Reperfusion with tPA: A Possible Role of TXNIP-NLRP3 Inflammasome.

OBJECTIVES: Acute hyperglycemia (HG) exacerbates reperfusion injury after stroke. Our recent studies showed that acute HG upregulates thioredoxin-interacting protein (TXNIP) expression, which in turn induces inflammation and neurovascular damage in a suture model of ischemic stroke. The aim of the present study was to investigate the effect of acute HG on TXNIP-associated neurovascular damage, in a more clinically relevant murine model of embolic stroke and intravenous tissue plasminogen activator (IV-tPA) reperfusion. MATERIALS AND METHODS: HG was induced in adult male mice, by intraperitoneal injection of 20% glucose. This was followed by embolic middle cerebral artery occlusion (eMCAO), with or without IV-tPA (10 mg/kg) given 3 h post embolization. Brain infarction, edema, hemoglobin content, expression of matrix metalloproteinase (MMP-9), vascular endothelial growth factor A (VEGFA), tight junction proteins (claudin-5, occluding, and zonula occludens-1), TXNIP, and NOD-like receptor protein3 (NLRP3)-inflammasome activation were evaluated at 24 h after eMCAO. RESULTS: HG alone significantly increased TXNIP in the brain after eMCAO, and this was associated with exacerbated hemorrhagic transformation (HT; as measured by hemoglobin content). IV-tPA in HG conditions showed a trend to decrease infarct volume, but worsened HT after eMCAO, suggesting that HG reduces the therapeutic efficacy of IV-tPA. Further, HG and tPA-reperfusion did not show significant differences in expression of MMP-9, VEGFA, junction proteins, and NLRP3 inflammasome activation between the groups. CONCLUSION: The current findings suggest a potential role for TXNIP in the occurrence of HT in hyperglycemic conditions following eMCAO. Further studies are needed to understand the precise role of vascular TXNIP on HG/tPA-induced neurovascular damage after stroke.

Inhibition of the PERK/TXNIP/NLRP3 Axis by Baicalin Reduces NLRP3 Inflammasome-Mediated Pyroptosis in Macrophages Infected with Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) remains a significant threat to global health as it induces granuloma and systemic inflammatory responses during active tuberculosis. Mtb can induce macrophage pyroptosis, leading to the release of IL-1ß and tissue damage, promoting its spread. Here, we established an in vitro Mtb-infected macrophage model to seek an effective antipyroptosis agent. Baicalin, isolated from Radix Scutellariae, was found to reduce pyroptosis in Mtb-infected macrophages. Baicalin could inhibit activation of the PERK/eIF2α pathway and thus downregulates TXNIP expression and subsequently reduces activation of the NLRP3 inflammasome, resulting in reduced pyroptosis in Mtb-infected macrophages. In conclusion, baicalin reduced pyroptosis by inhibiting the PERK/TXNIP/NLRP3 axis and might thus be a new adjuvant host-directed therapy (HDT) drug.

TXNIP contributes to bone loss via promoting the mitochondrial oxidative phosphorylation during glucocorticoid-induced osteoporosis.

Oxidative stress is a promoting factor in the pathologic process of glucocorticoid - induced osteoporosis (GIO), while the mechanism is still unclear. Thioredoxin-interacting protein (TXNIP) is a vital protein responsible for regulation of cellular reactive oxygen species (ROS) generation elicited by mitochondrial oxidative stress, and which may activate oxidative phosphorylation under the pathogenic status. In this research, the results showed that signaling pathway associated with the mitochondrial oxidative phosphorylation (MOP) down-regulated under conditions of TXNIP siRNA in MG63 cells. Furthermore, the evidence revealed that the expression level of TXNIP in serum and bone was elevated in a rat of GIO. Moreover, the differential proteins (Ndufs3, SDHD, Cyt B, COX IV, and ATP B) related to MOP pathway were identified to down-regulate in the proteomics of bone tissues by using isobaric Tags for Relative and Absolute Quantification (iTRAQ) method in TXNIP knockout mice treated with glucocorticoid, and the proteins were also verified by simple western blot. Taken together, the present findings highlights that TXNIP involves in triggering the process of bone loss via up-regulation of the MOP pathway, resulting to GIO, while TXNIP knockout can prevent the pathogenesis of GIO to some extent.

Structure basis of non-structural protein pA151R from African Swine Fever Virus.

African Swine Fever Virus (ASFV) is an enveloped double-stranded DNA icosahedral virus that causes the devastating hemorrhagic fever of pigs. ASFV infections severely impact swine production and cause an enormous economic loss, but no effective vaccine and therapeutic regimen is available. pA151R is a non-structural protein of ASFV, which is expressed at both early and late stages of viral infection. Significantly, pA151R may play a key role in ASFV replication and virus assembly as suppressing pA151R expression can reduce virus replication. However, little is known about the functional and structural mechanisms of pA151R because it shares a very low sequence identity to known structures. It was proposed that pA151R might participate in the redox pathway owing to the presence of a thioredoxin active site feature, the WCTKC motif. In this study, we determined the crystal structure of pA151R. Based on the crystal structure, we found that pA151R comprises of a central five-stranded ß-sheet packing against two helices on one side and an incompact C-terminal region containing the WCTKC motif on the other side. Notably, two cysteines in the WCTKC motif, an additional cysteine C116 from the ß7-ß8 loop together with ND1 of H109 coordinate a Zn2+ ion to form a Zn-binding motif. These findings suggest that the structure of pA151R is significantly different from that of typical thioredoxins. Our structure should provide molecular insights into the understanding of functional and structural mechanisms of pA151R from ASFV and shall benefit the development of prophylactic and therapeutic anti-ASFV agents.

Thioredoxin-1 attenuates atherosclerosis development through inhibiting NLRP3 inflammasome.

BACKGROUNDS: The thioredoxin-1 has atheroprotective effects via regulating oxidative stress and inflammation. In addition, the NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome also contributes to atherosclerosis development. However, whether the thioredoxin-1 suppresses atherosclerosis development by modulating the NLRP3 inflammasome remains unclear. METHODS: The regulation of NLRP3 inflammasome by thioredoxin-1 was determined in vitro on macrophage cells after ox-LDL (oxidized low-density lipoprotein) stimulation. The IL-1ß and caspase-1 p10 secretion were assessed by ELISA and western blot. Finally, the thioredoxin-1/NLRP3 inflammasome pathway was confirmed in apolipoprotein E-deficient mice. RESULTS: Thioredoxin-1 suppressed the expression of NLRP3, the secretion of IL-1ß and caspase-1 p10 in vitro. And ROS stimulation activated the NLRP3 inflammasome which was inhibited by thioredoxin-1. In the mouse model of atherosclerosis, thioredoxin-1 delivered by lentivirus vector inhibited atherosclerosis development. And the atheroprotective effects of thioredoxin-1 were attenuated by ROS stimulation. Furthermore, the regulation of NLRP3 inflammasome by thioredoxin-1 was also confirmed in vivo. CONCLUSIONS: We demonstrated here that the thioredoxin-1 had atheroprotective functions through thioredoxin-1/NLRP3 inflammasome pathway.

TXNIP deficiency mitigates podocyte apoptosis via restraining the activation of mTOR or p38 MAPK signaling in diabetic nephropathy.

Thioredoxin-interacting protein (TXNIP), is identified as an inhibitor of the thiol oxidoreductase thioredoxin that acts endogenously, and is increased by high glucose (HG). In this study, we investigated the potential function of TXNIP on apoptosis of podocytes and its potential mechanism in vivo and in vitro in diabetic nephropathy (DN). TXNIP silencing attenuated HG-induced apoptosis and obliterated the activation of signaling pathways of mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (MAPK) in conditionally immortalized mouse podocytes. Furthermore, the Raptor and Rictor shRNAs, mTOR specific inhibitor KU-0063794 and p38 MAPK inhibitor SB203580 were used to assess the role of mTOR or p38 MAPK pathway on podocyte apoptosis induced by HG. The Rictor and Raptor shRNAs and KU-0063794 appeared to reduce HG-induced apoptosis in podocytes. Simultaneously, SB203580 could also restrain HG-induced apoptosis in podocytes. Streptozotocin rendered equivalent diabetes in TXNIP-/- (TKO) and wild-type (WT) control mice. TXNIP deficiency mitigated renal injury in diabetic mice. Additionally, TXNIP deficiency also descended the apoptosis-related protein and Nox4 levels, the mTOR signaling activation and the p38 MAPK phosphorylation in podocytes of diabetic mice. All these data indicate that TXNIP deficiency may mitigate apoptosis of podocytes by inhibiting p38 MAPK or mTOR signaling pathway in DN, underlining TXNIP as a putative target for therapy.

Thioredoxin1 Inactivation Mediates the Impairment of Ischemia-Induced Angiogenesis and Further Injury in Diabetic Myocardium.

Diabetes mellitus (DM)-induced impairment of collateral formation has been demonstrated in subjects with coronary artery disease, which contributes to unfavorable prognosis among diabetic individuals. In our previous studies, thioredoxin1 (Trx1) activity was shown to be decreased in diabetic cardiac tissues, but the reason of Trx1 inactivation and whether it mediates the impaired angiogenesis in ischemic myocardium is still to be identified. As thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of Trx, is overexpressed in DM due to carbohydrate response element within its promoter, we hypothesized that inhibition of Trx1 by enhanced TXNIP expression in endothelial cells may play a role in hyperglycemia-induced impairment of angiogenesis. In the present study, we found that high glucose-mediated increase of TXNIP expression and TXNIP-Trx1 interaction induced the impairment in endothelial cell function and survival, since these detrimental effects are rescued by silencing TXNIP with small interfering RNA. In diabetic mice, TXNIP knockdown or recombinant human Trx1 treatment counteracted the impairment of angiogenesis, alleviated myocardial ischemic injury, and improved survival rate. All these data implicate that TXNIP upregulation and subsequently the increased formation of TXNIP-Trx1 complex is a novel pathologic pathway by which DM induces insufficient angiogenesis and thereby exacerbates myocardial ischemia injury.

Salvianolic acid A attenuated myocardial infarction-induced apoptosis and inflammation by activating Trx.

Myocardial infarction (MI) is a leading cause of mortality worldwide and it is urgent to discover effective therapies. In this study, the protective effect of salvianolic acid A (SAL) on MI induced by left anterior descending coronary artery ligation surgery and H2O2-induced H9c2 damage was evaluated. Rats were intraperitoneally injected with SAL once a day for 2 days before MI. At 24-h post-MI, the SAL-treated group showed significantly decreased infarct rate and enhanced myocardial function. Meanwhile, myocardial injury enzymes such as aspartate transaminase (AST), lactate dehydrogenase (LDH), and creatine kinase (CK) were significantly reduced by SAL treatment. Taking advantage of RNA-seq technology, 52 disease targets of MI were associated with differentially expressed genes after SAL treatment in MI, among which 21 inflammation-related genes and 16 MAPK cascade-related genes were found. Further experiment indicated that SAL treatment reduced inflammatory factors such as IL-1ß, IL-6, and TNF-α and decreased tunnel-positive cells and pro-apoptotic Bax after MI. Further investigation revealed that SAL treatment elevated thioredoxin (Trx) and inhibited the activation of c-jun N-terminal kinase (JNK) to attenuate apoptosis and inflammation after MI. Consistently, SAL protected cardiomyocytes against H2O2-induced H9c2 damage through increasing cell viability, decreasing cell apoptosis, and activating Trx and inhibiting JNK. Taken together, SAL inhibited cell apoptosis and inflammation through Trx/JNK signaling.

The tomato WV gene encoding a thioredoxin protein is essential for chloroplast development at low temperature and high light intensity.

BACKGROUND: Chloroplast biogenesis, a complex process in higher plants, is the key to photoautotrophic growth in plants. White virescent (wv) mutants have been used to unfold the molecular mechanisms underlying the regulation of chloroplast development and chloroplast gene expression in plants. However, most of genes controlling white virescent phenotype still remain unknown. RESULTS: In this study, we identified a temperature- and light intensity-sensitive mutant, named as wv. The content of chlorophyll was dramatically decreased in the immature leaves of wv mutant under the conditions of low temperature and high-light intensity. TEM observation showed that the chloroplasts in the young leaves of wv mutant lacked an organized thylakoid membrane, whereas crescent-shaped chloroplasts with well-developed stromal and stacked grana thylakoids in the mature leaves were developed. Immunoblot analyses suggested that proteins of photosynthetic complexes were decreased substantially in wv mutants. Based on map-based cloning and transgenic analysis, we determined that the wv phenotype was caused by single base mutation in the first intron of WV gene, which encoded a thioredoxin protein with 365 amino acids. qRT-PCR analysis revealed that the expression of WV gene was significantly down-regulated in wv mutant. In addition, knockdown of WV gene through RNAi also resulted in white virescent young leaves, suggesting that the mutation possibly blocks the differentiation of chloroplasts through inhibiting the expression of WV gene. Furthermore, the expression of WV peaked in apical buds and gradually decreased along with the developmental stage, which was consistent with the wv mutant phenotype. Expression analysis of chloroplast-encoded genes by qRT-PCR showed that the wv mutation affected the expression pattern of chloroplast-encoded PEP dependent genes. CONCLUSION: Our results suggested that wv mutant was sensitive to low temperature and light intensity. WV gene was essential for chloroplast differentiation. A single base mutation in the first intron resulted in down-regulation of WV gene expression, which inhibited the expression of chloroplast-encoded genes, thereby blocking chloroplast formation and chlorophyll synthesis.

Thioredoxins and thioredoxin reductase in chloroplasts: A review.

The chloroplastic thioredoxins (Trxs), a family of thiol-disulphide oxidoreductases, are reduced by either ferredoxin (Fd)-dependent Trx reductase (FTR) or reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent Trx reductase (NTR). Two Trx systems are present in chloroplasts including Trxs, Trx-like proteins, and reductase FTR and NTRC. FTR is the main reductant for Trxs in chloroplasts, while the flavoprotein NTRC integrates NTR and Trx activity, and plays multiple roles in the Calvin cycle, the oxidative pentose phosphate pathway (OPPP), anti-peroxidation, tetrapyrrole metabolism, ATP and starch synthesis, and photoperiodic regulation. In addition, not only there exists a reduction potential transfer pathway across the thylakoid membrane, but also FTR and NTRC coordinate with each other to regulate chloroplast redox homeostasis. Herein, we summarise the physiological functions of these two Trx reduction systems, discuss how both regulate redox homeostasis in plant plastids, and emphasize the significance of chloroplast thioredoxin systems in maintaining photosynthetic efficiency in plants.

Thioredoxin-mediated redox signalling in plant immunity.

Activation of plant immune responses is associated with rapid production of vast amounts of reactive oxygen and nitrogen species (ROS/RNS) that dramatically alter cellular redox homeostasis. Even though excessive ROS/RNS accumulation can cause widespread cellular damage and thus constitute a major risk, plant cells have evolved to utilise these molecules as important signalling cues. Particularly their ability to modify redox-sensitive cysteine residues has emerged as a key mechanism to control the activity, conformation, protein-protein interaction and localisation of a growing number of immune signalling proteins. Regulated reversal of cysteine oxidation is dependent on activities of the conserved superfamily of Thioredoxin (TRX) enzymes that function as cysteine reductases. The plant immune system recruits specific TRX enzymes that have the potential to functionally regulate numerous immune signalling proteins. Although our knowledge of different TRX immune targets is now expanding, little remains known about how these enzymes select their substrates, what range of oxidized residues they target, and if they function selectively in different redox-mediated immune signalling pathways. In this review we discuss these questions by examining evidence showing TRX enzymes exhibit novel activities that play important roles in diverse aspects of plant immune signalling.

The Thioredoxin-Like Family of Selenoproteins: Implications in Aging and Age-Related Degeneration.

The thioredoxin-like (Rdx) family proteins contain four selenoproteins (selenoprotein H, SELENOH; selenoprotein T, SELENOT; selenoprotein V, SELENOV; selenoprotein W, SELENOW) and a nonselenoprotein Rdx12. They share a CxxU or a CxxC (C, cysteine; x, any amino acid; U, selenocysteine) motif and a stretch of eGxFEI(V) sequence. From the evolutionary perspective, SELENOW and SELENOV are clustered together and SELENOH and SELENOT are in another branch. Selenoproteins in the Rdx family exhibit tissue- and organelle-specific distribution and are differentially influenced in response to selenium deficiency. While SELENOH is nucleus-exclusive, SELENOT resides mainly in endoplasmic reticulum and SELENOW in cytosol. SELENOV is expressed essentially only in the testes with unknown cellular localization. SELENOH and SELENOW are more sensitive than SELENOT and SELENOV to selenium deficiency. While physiological functions of the Rdx family of selenoproteins are not fully understand, results from animal models demonstrated that (1) brain-specific SELENOT knockout mice are susceptible to 1-methyl-4-phenylpyridinium-induced Parkinson's disease in association with redox imbalance and (2) adult zebrafishes with heterozygous SELENOH knockout are prone to dimethylbenzanthracene-induced tumorigenesis together with increased DNA damage and oxidative stress. Further animal and human studies are needed to fully understand physiological roles of the Rdx family of selenoproteins in redox regulation, genome maintenance, aging, and age-related degeneration.

TXNDC9 promotes hepatocellular carcinoma progression by positive regulation of MYC-mediated transcriptional network.

The thioredoxin domain containing proteins are a group of proteins involved in redox regulation and have been recently reported to be associated with tumor progression. However, the role of thioredoxin proteins in hepatocellular carcinoma (HCC) remains largely unknown. Here in our study, we demonstrated that thioredoxin domain containing protein 9 (TXNDC9) was over-expressed in HCC and promoted HCC progression. We found that TXNDC9 expression was amplified in HCC tissues and associated with an advanced grade of HCC. And, we demonstrated that overexpression of TXNDC9 was correlated with poor prognosis of HCC. Furthermore, by using CRISPR-Cas9 mediated TXNDC9 knockout and RNA-seq analysis, we found that TXNDC9 accelerated HCC proliferation regulation. Moreover, we demonstrated that TXNDC9 directly interacted with MYC and knockout/knockdown of TXNDC9 decreased the protein levels of MYC and inhibited MYC-mediated transcriptional activation of its targets. Besides, we identified that TXNDC9 was trans-activated by FOXA1, JUND, and FOSL2 in HCC. Taken together, our study unveiled an oncogenic role of TXNDC9 in HCC and provided a mechanistic insight into the TXNDC9 mediated gene regulation network during HCC development.

[Do Myokines Have Potential as Exercise Mimetics?]

　Exercise is generally considered to have health benefits for the body, although its beneficial mechanisms have not been fully elucidated. Recent progressive research suggests that myokines, bioactive substances secreted from skeletal muscle, play an important role in mediating the benefits of exercise. There are three types of myokines in terms of the muscular secretion mechanism: those in which the secretion is promoted by stimulation, such as irisin, interleukin (IL)-6, and IL-15; those whose secretion is constitutive, such as thioredoxin, glutaredoxin, and peroxiredoxin; and those whose secretion is suppressed by stimulation, such as by a macrophage migration inhibitory factor. Although dozens of myokines have been reported, their physiological roles are not well understood. Therefore, there currently exists no advanced drug discovery research specifically targeting myokines, with the exception of Myostatin. Myostatin was discovered as a negative regulator of muscle growth. Myostatin is secreted from muscle cells as a myokine; it signals via an activin type IIB receptor in an autocrine manner, and regulates gene expressions involved in myogenesis. Given the studies to date that have been conducted on the utilization of myostatin inhibitors for the treatment of muscle weakness, including cachexia and sarcopenia, other myokines may also be new potential drug targets.

Cellular stress and redox activity proteins are involved in gastric carcinogenesis associated with Helicobacter pylori infection expressing high levels of thioredoxin-1.

Helicobacter pylori infection is related to the development of gastric diseases. Our previous studies showed that high thioredoxin-1 (Trx1) expression in H. pylori can promote gastric carcinogenesis. To explore the underlying molecular mechanisms, we performed an isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis of stomach tissues from Mongolian gerbil infected with H. pylori expressing high and low Trx1. Differences in the profiles of the expressed proteins were analyzed by bioinformatics and verified using Western blot analysis. We found three candidate proteins, 14-3-3α/ß, glutathione-S-transferase (GST), and heat shock protein 70 (HSP70), in high Trx1 tissues compared with low Trx1 tissues and concluded that cellular stress and redox activity-related proteins were involved in the pathogenesis of gastric cancer associated with H. pylori Trx1.

[Effects and related mechanism of overexpression of human thioredoxin on the inflammatory response in mice with viral myocarditis].

Objective: To observe the effects of recombinant adenovirus with human thioredoxin (hTRX) on the inflammatory response in mice with viral myocarditis and explore the related mechanism. Methods: Sixty Balb/c male mice were randomly divided into control group, myocarditis group, and hTRX group according to the random number table (n=20 each group). The myocarditis group and hTRX group were injected with 100 TCID(50) Coxackie virus B3 (0.1 ml) in the abdomen and control group were injected with saline. Two days before the viral injection, the hTRX group were injected with recombinant adenovirus vector coding the human thioredoxin gene by pericardial puncture and the control group and myocarditis group were injected with recombinant adenovirus vector without coding gene by pericardial puncture, all these mice were killed and hearts were removed 7 days later. The morphology of myocardial tissue in each group was detected by HE staining and the ultrastructure changes by electron microscope. The protein expressions of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and NF-κB were detected by ELISA and Western blot. Immunohistochemical staining was performed to observe the protein expression levels of thioredoxin. Results: Necrosis of myocardial cells and a small amount of cell infiltration were found in the myocarditis group and necrosis and cell infiltration were significantly reduced in the hTRX group and no myocardial lesion was found in control group on HE stained sections. Electron microscope examination evidenced cell swelling and dissolved myofilament, vacuoles degeneration in mitochondria in the myocarditis group. These changes were significantly reduced in the hTRX group. There was no myocardial lesion in control group. The protein expression of TNF-α, IL-1ß and NF-κB were significantly upregulated in myocarditis group than in control group (all P<0.01). The protein expression of TNF-α, IL-1ß and NF-κB were significantly downregulated in hTRX group than in myocarditis group (all P<0.01). Immunohistochemical staining showed that protein expression of hTRX was higher in hTRX group than in myocarditis group (P<0.01). Conclusion: Recombinant adenovirus hTRX can attenuate cardiac injury in mice with acute myocarditis via inhibiting the inflammatory response and downregulating the expression of TNF-α, IL-1ß and NF-κB.

Maintaining Cone Function in Rod-Cone Dystrophies.

Retinal degenerative diseases are a major cause of untreatable blindness due to a loss of photoreceptors. Recent advances in genetics and gene therapy for inherited retinal dystrophies (IRDs) showed that therapeutic gene transfer holds a great promise for vision restoration in people with currently incurable blinding diseases. Due to the huge genetic heterogeneity of IRDs that represents a major obstacle for gene therapy development, alternative therapeutic approaches are needed. This review focuses on the rescue of cone function as a therapeutic option for maintaining central vision in rod-cone dystrophies. It highlights recent developments in better understanding the mechanisms of action of the trophic factor RdCVF and its potential as a sight-saving therapeutic strategy.

The thioredoxin-1 system is essential for fueling DNA synthesis during T-cell metabolic reprogramming and proliferation.

The thioredoxin-1 (Trx1) system is an important contributor to cellular redox balance and is a sensor of energy and glucose metabolism. Here we show critical c-Myc-dependent activation of the Trx1 system during thymocyte and peripheral T-cell proliferation, but repression during T-cell quiescence. Deletion of thioredoxin reductase-1 (Txnrd1) prevents expansion the CD4-CD8- thymocyte population, whereas Txnrd1 deletion in CD4+CD8+ thymocytes does not affect further maturation and peripheral homeostasis of αßT cells. However, Txnrd1 is critical for expansion of the activated T-cell population during viral and parasite infection. Metabolomics show that TrxR1 is essential for the last step of nucleotide biosynthesis by donating reducing equivalents to ribonucleotide reductase. Impaired availability of 2'-deoxyribonucleotides induces the DNA damage response and cell cycle arrest of Txnrd1-deficient T cells. These results uncover a pivotal function of the Trx1 system in metabolic reprogramming of thymic and peripheral T cells and provide a rationale for targeting Txnrd1 in T-cell leukemia.

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

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 ß1 and ECM proteins in vivo. TXNDC5 mRNA and protein were increased in human CF (hCF) under transforming growth factor ß1 stimulation in vitro. Knockdown of TXNDC5 attenuated transforming growth factor ß1-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 ß1-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 ß 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.