Leashing Ferroptosis in Myocardial Ischemia/Reperfusion Injury: Role of the YAP/NEDD4L/ACSL4 Pathway, Knowledge Gaps, and Potential Therapeutic Application

Coronary artery disease is the leading cause of death in the world, causing serious complications such as myocardial infarction (MI). MI is followed by an ischemia-induced myocardial injury and then by a progressive remodelling of the heart, resulting in heart failure. Reperfusion is a definitive treatment for acute MI. However, by itself, reperfusion induces an additional myocardial injury, a process termed ischemia/reperfusion (I/R) injury. Despite an improved understanding of the pathophysiology of I/R injury, effective clinical therapies to prevent myocardial I/R injury remain limited, representing substantial gaps in patient care. Cardiomyocyte death is the key mechanism contributing to myocardial I/R injury, as it is a fundamental pathologic process that triggers the cascade of adverse myocardial remodelling, leading to heart failure.1Del Re D.P. Amgalan D. Linkermann A. Liu Q. Kitsis R.N. Fundamental mechanisms of regulated cell death and implications for heart disease.Physiol Rev. 2019; 99: 1765-1817Crossref PubMed Scopus (482) Google Scholar Because of an extremely limited capacity to regenerate cardiomyocytes, inhibition of cardiomyocyte death has been an attractive strategy to reduce cardiac pathology and prevent the development and progression of heart failure after MI. Different forms of cardiomyocyte death have been reported in I/R injury including apoptosis, necroptosis, pyroptosis, ferroptosis, and autophagy. Ferroptosis has recently received great attention in basic and clinical research.2Zhao W.K. Zhou Y. Xu T.T. Wu Q. Ferroptosis: opportunities and challenges in myocardial ischemia-reperfusion injury.Oxid Med Cell Longev. 2021; 20219929687Google Scholar Ferroptosis is an iron-dependent form of programmed cell death characterized by increased lipid peroxidation and a decompensated antioxidant system. It is positively regulated by transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), acyl-CoA synthase long-chain family member 4 (ACSL4), lysophosphatidylcholine acyltransferase 3 (LPCAT3), and heme oxygenase 1 (HMOX1) and negatively regulated by glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), the nuclear factor erythroid 2-related factor 2 (NRF2), and ferroptosis suppressor protein 1 (FSP1).3Sheng S.-Y. Li J.-M. Hu X.-Y. Wang Y. Regulated cell death pathways in cardiomyopathy.Acta Pharm Sin. 2023; 44: 1521-1535Crossref PubMed Scopus (4) Google Scholar It is known that myocardial I/R injury induces iron accumulation and increased lipid peroxidation alongside a reduction of GPX4 and an increase in ACSL4 in preclinical animal models, strongly supporting the occurrence of ferroptosis. Notably, administration of ferroptosis inhibitors (ferrostatin-1, liproxstatin-1, and deferoxamine) or transgenic overexpression of GPX4 reduces myocardial I/R injury and improves myocardial function, highlighting an important role of ferroptosis in myocardial I/R injury.2Zhao W.K. Zhou Y. Xu T.T. Wu Q. Ferroptosis: opportunities and challenges in myocardial ischemia-reperfusion injury.Oxid Med Cell Longev. 2021; 20219929687Google Scholar Thus, targeting ferroptosis regulatory mechanisms may represent a useful therapy to reduce myocardial I/R injury and prevent its progression to heart failure after MI.4Yang X. Kawasaki N.K. Min J. Matsui T. Wang F. Ferroptosis in heart failure.J Mol Cell Cardiol. 2022; 173: 141-153Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar However, research on ferroptosis is still in its infancy, and ferroptosis regulatory mechanisms have not yet been fully addressed in myocardial I/R injury. In this issue of the Canadian Journal of Cardiology, Qiu et al.5Qiu M. Yan W. Liu M. YAP facilitates NEDD4L-mediated ubiquitination and degradation of ACSL4 to alleviate ferroptosis in myocardial ischemia-reperfusion injury.Can J Cardiol. 2023; 39: 1712-1727Abstract Full Text Full Text PDF Scopus (1) Google Scholar investigated the role of the YAP/NEDD4L1/ACSL4 pathway in ferroptosis during myocardial I/R injury to address this knowledge gap. They reported that YAP promotes NEDD4L expression by interacting with TEA domain family member-4 (TEAD4), a transcription factor contributing to NEDD4L transcription in cardiomyocytes following oxygen-glucose deprivation/reoxygenation, an in vitro condition simulating I/R. NEDD4L belongs to the NEDD4 family, the largest group of ubiquitin E3 ligases, which play an important role in post-translational modifications (PTMs) of proteins through ubiquitination. In general, ubiquitination of proteins leads to either degradation or activation of the modified proteins, thus regulating the biological function mediated by these proteins. Protein ubiquitination is a cascade reaction mediated by 3 types of enzymes including ubiquitin-activating enzyme (E1), ubiquitin cross-linking enzyme (E2), and ubiquitin ligase (E3). NEDD4L has been recognized to be a crucial regulator of hypertension, myocardial infarction, and arrhythmias.6Li M. Sun G. Wang P. et al.Research progress of Nedd4L in cardiovascular diseases.Cell Death Discov. 2022; 8: 206Crossref PubMed Scopus (11) Google Scholar Qiu et al. further revealed that NEDD4L targets acyl-CoA synthetase long-chain family member 4 (ACSL4), an inducer of ferroptosis7Yuan H. Li X. Zhang X. Kang R. Tang D. Identification of ACSL4 as a biomarker and contributor of ferroptosis.Biochem Biophys Res Commun. 2016; 478: 1338-1343Crossref PubMed Scopus (550) Google Scholar and promotes its ubiquitination and degradation, which eventually inhibits ferroptosis in cardiomyocytes following oxygen-glucose deprivation/reoxygenation.5Qiu M. Yan W. Liu M. YAP facilitates NEDD4L-mediated ubiquitination and degradation of ACSL4 to alleviate ferroptosis in myocardial ischemia-reperfusion injury.Can J Cardiol. 2023; 39: 1712-1727Abstract Full Text Full Text PDF Scopus (1) Google Scholar These findings demonstrate a new regulatory mechanism contributing to ferroptosis in cardiomyocytes. To provide in vivo insights into the role of the YAP/NEDD4L1/ACSL4 pathway, Qiu et al. showed that overexpression of YAP and NEDD4L in a rat model of myocardial I/R injury attenuates cardiac ferroptosis, reduces myocardial injury, and improves myocardial function,5Qiu M. Yan W. Liu M. YAP facilitates NEDD4L-mediated ubiquitination and degradation of ACSL4 to alleviate ferroptosis in myocardial ischemia-reperfusion injury.Can J Cardiol. 2023; 39: 1712-1727Abstract Full Text Full Text PDF Scopus (1) Google Scholar suggesting that targeting the YAP/NEDD4L1/ACSL4 pathway may be a therapeutic approach to reduce myocardial I/R injury. Thus, the study by Qiu et al. highlighted a novel role of YAP/NEDD4L1/ACSL4 pathway in myocardial I/R injury by modulating ferroptosis. However, there are some limitations in this study by Qiu et al. First, the direct evidence of ferroptosis in myocardial I/R injury is still lacking, as most biomarkers of ferroptosis are also present in other forms of regulated cell death or pathologic conditions.8Chen X. Comish P.B. Tang D. Kang R. Characteristics and biomarkers of ferroptosis.Front Cell Dev Biol. 2021; 9637162Google Scholar Therefore, more specific biomarkers for ferroptosis warrant further investigations. This is also a major challenge in the field. Second, different forms of cardiomyocyte death have been reported during myocardial I/R injury. So, what are the relative contributions of ferroptosis vs nonferroptotic cell death to myocardial I/R injury? Third, the incidence of ferroptosis may be not the same at different stages of myocardial I/R injury and its progression to heart failure. So, at which stage of myocardial I/R injury does ferroptosis take place? Fully addressing these limitations will provide strong rationales for targeting ferroptosis regulatory mechanisms as a potential therapy to reduce myocardial I/R injury. To provide further molecular insights, it will be interesting to explore how NEDD4L targets and ubiquitinates ACSL4. This could include 3 aspects: subcellular localization of the interaction between NEDD4L and ACSL4; the ubiquitination type of ACSL4 induced by NEDD4L; and the detailed residue in ACSL4, which is ubiquitinated by NEDD4L. NEDD4L has been reported to ubiquitinate many membrane proteins, including epithelial sodium channel (ENaC).9Staub O. Gautschi I. Ishikawa T. et al.Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination.EMBO J. 1997; 16: 6325-6336Crossref PubMed Scopus (608) Google Scholar Therefore, it is of interest to know if the NEDD4L-ACSL4 interaction occurs near the cell membrane, which could further affect cell membrane lipid peroxidation and ferroptosis. In addition, ubiquitin has 7 internal lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63), each of which could conjugate with the C-terminus of another ubiquitin molecule to form a type of polyubiquitination chain for the conjugation of protein substrates.10Zhao Q. Zhang R. Qiao C. Miao Y. Yuan Y. Zheng H. Ubiquitination network in the type I IFN-induced antiviral signaling pathway.Eur J Immunol. 2023; 53e2350384Crossref Scopus (1) Google Scholar Among them, the Lys48 residue forms a Lys48 (K48)-linked polyubiquitination chain that, in general, leads to the degradation of the modified substrate proteins. However, whether or not NEDD4L induces K48-linked polyubiquitination of ACSL4 remains to be explored. Moreover, identification of the lysine residue in ACSL4 protein that is modified by the polyubiquitination chain mediated by NEDD4L could provide significant insights into NEDD4L regulation of ACSL4 stability. It is important to mention that NEDD4L has been recently reported to promote ferroptosis in tumour cells through ubiquitination and degradation of GPX4,11Cheng F. Dou J. Yang Y. et al.Drug-induced lactate confers ferroptosis resistance via p38-SGK1-NEDD4L-dependent upregulation of GPX4 in NSCLC cells.Cell Death Discov. 2023; 9: 165Crossref PubMed Scopus (2) Google Scholar,12Tang H. Jiang X. Hua Y. et al.NEDD4L facilitates granulosa cell ferroptosis by promoting GPX4 ubiquitination and degradation.Endocr Connect. 2023; 12e220459Crossref Scopus (2) Google Scholar suggesting that the role of NEDD4L in ferroptosis may be cell-type dependent. In addition to NEDD4L, other ubiquitin E3 ligases have been reported to negatively or positively regulate ferroptosis by targeting SLC7A11, GPX4, Chac1, NRF2, and so forth,13Meng Y. Sun H. Li Y. et al.Targeting ferroptosis by ubiquitin system enzymes: a potential therapeutic strategy in cancer.Int J Biol Sci. 2022; 18: 5475-5488Crossref PubMed Scopus (12) Google Scholar Interestingly, another ubiquitin E3 ligase, F-box only protein 10 (FBXO10), can ubiquitinate and degrade ACSL4, thereby regulating lipid peroxidation and ferroptosis.14Bao Z. Liu Y. Chen B. et al.Prokineticin-2 prevents neuronal cell deaths in a model of traumatic brain injury.Nat Commun. 2021; 12: 4220Crossref PubMed Scopus (41) Google Scholar Thus, it will be plausible to study how the cells differentially employ FBXO10-ACSL4, NEDD4L-ACSL4, or NEDD4L-GPX4 signalling for ferroptosis under different conditions. In summary, Qiu et al.5Qiu M. Yan W. Liu M. YAP facilitates NEDD4L-mediated ubiquitination and degradation of ACSL4 to alleviate ferroptosis in myocardial ischemia-reperfusion injury.Can J Cardiol. 2023; 39: 1712-1727Abstract Full Text Full Text PDF Scopus (1) Google Scholar provided concise evidence demonstrating that the YAP/NEDD4L1/ACSL4 pathway is a new regulatory mechanism for ferroptosis in myocardial I/R injury and may represent useful therapeutic targets to inform future translational/clinical studies and development of new drugs to reduce myocardial I/R injury. Nevertheless, the ferroptosis regulatory mechanisms are far more complicated than we know. Thus, a full understanding of ferroptosis merits further investigation before moving forward to future translational studies and clinical trials. No funding was provided for this paper.