Highlights
- S-nitrosylation (SNO) of Pyruvate Kinase Isoform 2 (PKM2) at Cys49 and Cys326 is a specific hallmark of activated cardiac fibroblasts in heart failure.
- SNO-PKM2 disrupts PKM2 tetramerization and reduces enzyme activity, shifting the metabolic and structural state of the cell.
- Mechanistically, SNO-PKM2 promotes excessive mitochondrial fission by interfering with the actin regulatory protein gelsolin, leading to mitochondrial dysfunction and fibroblast activation.
- The FDA-approved drug mitapivat and the activator TEPP-46 effectively reverse these pathways, presenting a viable therapeutic strategy for mitigating cardiac fibrosis.
Background
Cardiac fibrosis—the excessive deposition of extracellular matrix (ECM) proteins by activated fibroblasts—is a ubiquitous feature of chronic heart disease and a primary driver of heart failure (HF) progression. Despite its clinical significance, there are currently no approved therapies that directly and specifically target the activation of cardiac fibroblasts. Standard-of-care treatments, such as ACE inhibitors and beta-blockers, focus primarily on hemodynamics and neurohormonal pathways rather than the underlying fibrotic process.
Nitric oxide (NO) is a critical signaling molecule in the cardiovascular system, but under chronic pathological stress, excessive NO production leads to nitrosative stress. One major post-translational modification resulting from this stress is S-nitrosylation (SNO), the covalent attachment of an NO moiety to specific cysteine thiols. While SNO has been studied in the context of cardiomyocyte contractility, its role in the fibrotic transformation of cardiac fibroblasts remained largely undefined until the recent landmark study published in Circulation (Luo et al., 2026).
Key Content
Identification of SNO-PKM2 in Fibrogenesis
Through sophisticated SNO-proteomic analysis conducted on cardiac tissue from transverse aortic constriction (TAC) mice and spontaneous hypertensive rats (SHR), researchers identified Pyruvate Kinase M2 (PKM2) as a major target of S-nitrosylation. Notably, this modification was found to be highly enriched in cardiac fibroblasts but was almost absent in cardiomyocytes, suggesting a cell-specific role for SNO-PKM2 in the heart’s response to injury.
Validation in human samples further confirmed the clinical relevance of these findings. Heart tissue from patients with advanced heart failure exhibited significantly elevated levels of SNO-PKM2 at the cysteine 49 (Cys49) and cysteine 326 (Cys326) residues. This indicates that the SNO-PKM2 pathway is not merely a preclinical observation but a conserved pathological mechanism in human cardiac disease.
Biochemical and Metabolic Consequences of PKM2 S-Nitrosylation
PKM2 typically exists in a dynamic equilibrium between dimers and tetramers. The tetrameric form is enzymatically active and essential for standard glycolysis, whereas the dimeric form has lower pyruvate kinase activity and can translocate to the nucleus or engage in non-metabolic signaling.
The study by Luo et al. demonstrated that S-nitrosylation at Cys49 and Cys326 promotes the dissociation of PKM2 tetramers into dimers. This biochemical shift significantly reduces pyruvate kinase activity. In cardiac fibroblast-specific PKM2 knockout mice, the loss of PKM2 exacerbated fibrosis, suggesting that functional PKM2 is necessary to maintain fibroblast quiescence. Conversely, the introduction of a SNO-resistant PKM2 mutant (where Cys49 and Cys326 were mutated to prevent S-nitrosylation) successfully protected against fibrosis and preserved cardiac function in pressure-overload models.
The Mechanistic Bridge: Mitochondrial Fission and Gelsolin
One of the most profound insights from this research is the link between SNO-PKM2 and mitochondrial dynamics. Excessive mitochondrial fission—the fragmentation of mitochondria—is a known driver of cellular activation and oxidative stress in various disease contexts.
Through unbiased proteomics and co-immunoprecipitation (Co-IP) coupled with mass spectrometry, the researchers discovered that SNO-PKM2 interferes with the interaction between PKM2 and the actin-regulatory protein, gelsolin. In a healthy state, PKM2 interacts with gelsolin to maintain mitochondrial homeostasis. However, when PKM2 is S-nitrosylated, this interaction is disrupted, leading to the recruitment of fission-related proteins (such as Drp1) to the mitochondrial membrane. This results in excessive mitochondrial fragmentation, impaired bioenergetics, and the subsequent activation of the fibroblast into a pro-fibrotic myofibroblast phenotype.
Pharmacological Reversal: From TEPP-46 to Mitapivat
Given that SNO-PKM2 drives fibrosis by destabilizing the PKM2 tetramer, the researchers investigated whether pharmacological stabilizers could reverse this effect.
1. **TEPP-46:** This small-molecule PKM2 activator was shown to promote tetramerization, effectively alleviating mitochondrial fission and reducing the expression of fibrotic markers (such as Collagen I and alpha-SMA) in activated fibroblasts.
2. **Mitapivat:** More significantly, the study explored mitapivat, a drug recently approved by the US FDA for the treatment of hemolytic anemia in patients with pyruvate kinase deficiency. Mitapivat acts as a potent allosteric activator of PKM2. In experimental models, mitapivat showed both preventive and therapeutic efficacy, dose-dependently relieving cardiac fibrosis and improving ejection fraction. This highlights a clear opportunity for drug repurposing in cardiovascular medicine.
Expert Commentary
The discovery of the SNO-PKM2-gelsolin-mitochondria axis represents a paradigm shift in our understanding of cardiac fibrosis. Most previous research focused on TGF-beta signaling or metabolic reprogramming (the Warburg effect) in fibroblasts. By identifying S-nitrosylation as the ‘trigger’ for metabolic and structural dysfunction, this study provides a more precise target for intervention.
The cell-specific nature of SNO-PKM2 is particularly promising. A recurring challenge in developing anti-fibrotic drugs is the risk of off-target effects in cardiomyocytes or other organs. The fact that SNO-PKM2 is predominantly found in activated fibroblasts suggests that PKM2 activators may have a high therapeutic index with minimal adverse effects on healthy heart muscle.
From a clinical perspective, the transition of mitapivat from a rare blood disorder treatment to a potential heart failure therapy is an exciting development. Because mitapivat has already passed rigorous safety and pharmacology testing for its current indication, the pathway to Phase II clinical trials for cardiac fibrosis could be significantly shortened. However, clinicians must remain cautious; while the preclinical data is robust, the translation from murine TAC models to the heterogeneous landscape of human heart failure (HFrEF vs. HFpEF) requires careful patient stratification.
Conclusion
The research led by Luo and colleagues establishes S-nitrosylated PKM2 as a critical driver of cardiac fibrosis. By promoting the dissociation of PKM2 tetramers, nitrosative stress triggers a cascade of mitochondrial fission mediated by gelsolin, ultimately transforming quiescent fibroblasts into pathological myofibroblasts. The ability of mitapivat to stabilize PKM2 and arrest this process offers a compelling new therapeutic avenue. Future research should focus on the long-term safety of PKM2 activation in chronic cardiac conditions and explore the synergy between PKM2 activators and existing standard-of-care treatments for heart failure.
References
- Luo S, Ye D, Zhang Y, et al. S-Nitrosylation of Pyruvate Kinase Isoform 2 Drives Cardiac Fibrosis by Promoting Mitochondrial Fission. Circulation. 2026;153(5):338-357. PMID: 41368700.
- Hansen J, et al. Mitochondrial dynamics in the heart: The role of fission and fusion in fibrosis and failure. J Mol Cell Cardiol. 2021;158:12-24.
- Raimundo N, et al. Metabolic reprogramming of fibroblasts in pathological fibrosis. Trends Cell Biol. 2022;32(11):950-963.
