Previous studies have highlighted the role of the SARS-CoV-2 spike (S) protein in cardiovascular pathology. Evidence suggests that the S protein can independently induce cardiovascular complications by binding to cell membrane receptors, leading to inflammation and damage to cells, tissues, and organs. In this study, the authors from Taiwan investigated the effects of SARS-CoV-2 S protein on cultured human cardiac fibroblasts (CFs) and the molecular mechanisms underlying cardiac fibrosis induced by SARS-CoV-2 S protein.
Cardiovascular complications, including myocardial injury, heart failure, arrhythmias, and coagulation disorders, can manifest not only during the acute phase of COVID-19 but also in long-COVID-19 or post-acute COVID-19 (PACS) syndrome. Several previous studies have shown signs of non-ischemic myocardial fibrosis in patients diagnosed with PASC, exceeding its prevalence in the normal adult population. https://discovermednews.com/non-ischemic-myocardial-fibrosis-in-long-covid-patients/
The present study’s authors have noted that Cao et al. discovered that the SARS-CoV-2 S protein promotes cardiac fibrosis in obese mice, suggesting that the S protein caused myocardial contractile impairment by inducing long-term aberrances in the cardiac transcriptional signatures of mitochondrial respiratory chain genes. (Mol. Metab. 2023, 74, 101756) https://www.sciencedirect.com/science/article/pii/S221287782300090X?via%3Dihub
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped, positive-sense, single-stranded RNA virus. Its genome encodes four structural proteins, namely the spike (S), envelope (E), nucleocapsid (N), and membrane (M) protein. The S protein, composed of subunits S1 and S2, appears to be a major pathogenic factor contributing to the unique pathogenesis of SARS-CoV-2. The receptor binding domain (RBD) in the S1 subunit recognizes human ACE2 and is responsible for attachment to host cells.
The NOD-, LPR-, and pyrin-domain-containing protein 3 (NLRP3) is a component of intracellular pattern recognition receptors that detects a broad range of microbial motifs, endogenous danger signals, and environmental irritants, resulting in the formation and activation of NLRP3 inflammasome and release of the proinflammatory cytokines interleukin (IL)-1β and IL-18. Toll-like receptor 4 (TLR4) belongs to pattern recognition receptors that recognize conserved pathogen-associated molecular patterns, thus, representing the first line of defense against infection. It plays a pivotal role in the immune response to bacterial lipopolysaccharide (LPS) and other danger signals. Previous studies have shown that the S protein interacts with and activates TLR4 and its downstream signaling, triggering inflammatory responses in immune cells, the lungs, and the brain. The S protein can also activate the NLRP3 inflammasome in LPS-primed microglia through nuclear factor kappa-B (NF-κB) signaling in an angiotensin-converting enzyme 2 receptor (ACE2)-dependent manner. NF-κB can regulate the expression of hundreds of genes involved in inflammation, immunity, proliferation, and cell death.
The exact mechanisms involved in cardiac fibrosis, including the activation of CFs, as crucial cells, and the interaction of the S protein with NLRP3 and TLR4, remain poorly understood. The TLR4 and NLRP3 inflammasomes are highly involved in cardiac fibrosis development in response to inflammatory signals and pathogens. In addition, the S protein-induced TLR4 activation can trigger NLRP3 inflammasome activation. Therefore, the authors hypothesized that modulation of NLRP3 and TLR4 activity could attenuate the SARS-CoV-2 S protein-induced inflammatory response and associated cardiac injury.
Cardiac fibrosis
About the study
Researchers employed various methodologies, including scratch assays, Western blotting, and immunofluorescence, to assess the migration, fibrosis signaling, mitochondrial calcium levels, reactive oxygen species (ROS) production, and cell morphology of cultured human CFs treated with the S1 protein for 24 hours with or without an anti-ACE2 neutralizing antibody, a TLR4 blocker, or an NLRP3 inflammasome inhibitor. Human atrial fibroblasts were used as these cells exhibit greater secretory activity and reactivity, making them more suitable for studying the mechanisms of cardiac fibrosis.
The impact of S1 protein on the activation of CFs was assessed by scratch assay. The mechanisms by which the S1 protein activates CFs were analyzed through the expression of transforming growth factor β1 (TGF-β1) at both, the protein and mRNA levels.
The roles of NLRP3 and TLR4 in activating CFs were investigated by treating cells with an NLRP3 inflammasome inhibitor, MCC950, and a TLR4 inhibitor, TAK-242. The involvement of NF-κB in activating CFs by the S1 protein was investigated by treating cells with an NF-κB inhibitor, Bay 11-7082. The authors also assessed the activation of IL-1β, a marker of NF-κB and NLRP3 inflammasome activation. To investigate whether ACE2 receptors mediate the effects of the S1 protein on CFs, the cells were treated with an anti-ACE2 antibody before adding the S1 protein.
Results
The expression of myofibroblast markers was examined in a scratch assay, which revealed that exposure to the S1 protein significantly enhanced migration, but not the proliferation of human CFs.
The S1 protein enhanced expressions of collagen 1, α-smooth muscle actin, TGF-β1, phosphorylated SMAD2/3 (the downstream targets of TGF-β1), IL-1β, and NF-κB. These results indicate that S1 protein directly induced the expression of key fibrotic markers, playing a crucial role in promoting fibrotic processes within cardiac tissues. The S1 protein enhanced ROS production, however, it did not affect mitochondrial morphology or mitochondrial calcium content.
An NLRP3 inhibitor (MCC950) completely inhibited the effects of the S1 protein on CFs. It prevented the migration of CFs and overexpression of collagen 1, TGF-β1, and phosphorylated SMAD2/3, induced by S1 protein. Similarly, an NF-κB inhibitor (Bay 11-7082) effectively prevented the migration of CFs and suppressed the increase in expression of pro-COL1A1, TGF-β1, and IL-1β, induced by S1 protein. According to the authors, these results show that NF-κB mediates the CF activation induced by the S1 protein. The BAY 11-7082 also reversed the effect of the S1 protein on SMAD2/3 phosphorylation.
In contrast, the TLR4 inhibitor, TAK-242 did not exhibit inhibitory effects on these processes.
Treatment with an anti-ACE2 neutralizing antibody effectively attenuated the effects of the S1 protein on collagen 1 and TGF-β1 expression and prevented the CFs migration enhanced by the S1 protein, suggesting that observed effects of the S1 protein are ACE2-dependent.
Original illustration from Van Tin H, et al. Cells 2024, 13, 1331.
Conclusion
This study showed that the S1 protein activates human CFs by priming NLRP3 inflammasomes through NF-κB signaling in an ACE2-dependent way. Researchers emphasized that the molecular mechanisms underlying the effects of the S1 protein on CFs and the development of cardiac fibrosis are as follows: the S1 protein binds to ACE2, activating intracellular NF-κB signaling. The accumulation of ROS triggers activation of NLRP3 inflammasome and IL-1β production. Subsequently, IL-1β binds to its receptors, initiating signaling cascades that upregulate the transcription of TGF-β1, ultimately leading to cardiac fibrosis.
These results highlight the critical roles of NF-κB and NLRP3 inflammasome signaling pathways in the profibrotic effects of S1 protein in CFs and point to NF-κB signaling in the cellular response to SARS-CoV-2. Given the known role of NF-κB in regulating inflammatory and fibrotic processes, the authors suggested that targeting this signaling pathway may offer promising therapeutic approaches to prevent or treat cardiac fibrosis associated with COVID-19.
This article was published in Cells.
Journal Reference
Van Tin H, et al. Spike Protein of SARS-CoV-2 Activates Cardiac Fibrogenesis through NLRP3 Inflammasomes and NF-κB Signaling. Cells 2024, 13, 1331. https://doi.org/10.3390/cells13161331
