Cleavage of The Osmotic Stress‐Related Transcriptional Regulator NFAT5 Is Critical for Cardiac Injury in Coxsackievirus B3‐Induced Myocarditis
Bibliographic record
Abstract
Background Viral myocarditis is an inflammatory heart disease featured by severe cardiomyocyte death due to viral infection. Among different viruses, Coxsackievirus B3 (CVB3) is one of the most common causal agents of viral myocarditis. CVB3‐expressed proteases 2A and 3C can cleave cellular proteins, leading to acute damage to the heart. However, the mechanism underlying CVB3‐induced cardiac injury has not been well studied yet. Nuclear factor‐activated T cell 5 (NFAT5), a member of NFAT family, was originally identified as a transcriptional factor involved in hypertonic stresses. Recently, it was reported that NFAT5 plays a critical role in doxorubicin‐induced cytotoxicity for cardiac myocytes, in which degradation of NFAT5 induced by doxorubicin treatment remarkably reduces cell viability of cardiomyocytes. Besides, NFAT5 also represses the Wnt/β‐catenin signaling pathway by directly binding to β‐catenin and inhibiting the β‐catenin‐p300/CBP interaction, while high level of Wnt signals promotes cardiac fibrosis, decreases cardiac progenitor self renewal and activates cardiac inflammation, which are commonly observed in the progression of viral myocarditis. Thus, NFAT5 seems to play a protective role for cardiomyocytes to prevent cardiac injury; however whether this occurs in CVB3‐induced viral myocarditis has not been studied. Hypothesis NFAT5 is cleaved by viral proteases during CVB3 infection in cardiomyocytes, which enhances cell death and cardiac injury via promoting Wnt/β‐catenin signaling pathway. Methods Heart sections collected from CVB3‐infected mice were subjected to immunohistochemical (IHC) staining using antibodies targeting NFAT5 and CVB3 capsid protein VP1. The cleavage of NFAT5 in CVB3 infection was verified in CVB3‐infected cell culture and mouse heart tissue by Western blots (WB). The potential cleavage sites of CVB3 proteases 2A and 3C on NFAT5 were predicted by bioinformatic programs NetPicoRNA 1.0 and verified by site‐directed mutagenesis. The effects of NFAT5 on CVB3 replication and cell viability after infection was estimated by detecting VP1 synthesis and MTS assay, respectively. The activation of Wnt/β‐catenin signaling pathway was evaluated by TOPflash reporter activity and the binding between NFAT5 and β‐catenin was detected by immunoprecipitation. Results IHC staining showed no significant change of NFAT5 protein level in cardiomyocytes with or without CVB3 infection. Western blot analysis demonstrated that the 200‐kDa NFAT5 was cleaved after CVB3 infection to produce a 75 kDa N‐terminal fragment. Ectopic expression of 2A or 3C protease showed that CVB3 2A, but not 3C, is responsible for NFAT5 cleavage. Bioinformatic analysis predicted two glycine sites, G503 and G650, as the most potential cleavage sites, which were tested by sited‐directed mutagenesis and finally confirmed that G503 was the cleavage site. Functional study by expressing uncleavable NFAT5 mutant in cardiomyocytes demonstrated that uncleavable NFAT5 increased cell viability by 100% and reduced the synthesis VP1 by 60% during CVB3 infection. Immunoprecipitation and TOPflash reporter assay showed that cleaved NFAT5 could not bind to β‐catenin and promoted Wnt/β‐catenin signaling pathway. Conclusion NFAT5 is cleaved at G503 and inactivated by CVB3 protease 2A during infection, which enhances cardiomyocyte death and Wnt/β‐catenin signaling pathway, contributing to cardiac injury and viral pathogenesis. Support or Funding Information This work was supported by a grant from the Canadian Institutes of Health Research (grant # MOP‐125995).
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.001 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".