Targeting Nrf3-Pitx2-ROS axis represents a promising therapeutic strategy for myocardial infarction

Acute myocardial infarction (MI) claims millions of lives worldwide annually and remains the primary driver of chronic heart failure (HF). A hallmark of MI-induced cardiac damage is the extensive death of cardiomyocytes (CMs). Cardiac injury triggers massive mitochondria-derived reactive oxygen species (ROS), which directly or indirectly induce CM death, thereby contributing to cardiac damage during the acute phase of ischemia-reperfusion (I/R) injury and the chronic myocardial remodeling process after MI [1]. Consequently, inhibiting excessive mitochondrial ROS generation during cardiac injury has been suggested as an effective strategy to mitigate CM death and improve post-MI cardiac function. However, the lack of pharmacological agents capable of specifically inhibiting mitochondrial ROS generation in damaged hearts highlights an urgent clinical need.

Nuclear factor erythroid 2-related factor 3 (Nrf3) is a member of the Cap'n' collar-basic leucine zipper family of transcription factors, which also includes Nrf1 and Nrf2 [2]. Nrf1 functions as a highly-conserved determinant that sustains robust redox homeostasis in the eco-evo-devo processes of life histories [3]. Nrf2 is well established as a positive regulator of constitutive and inducible expression of numerous cytoprotective genes, including NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione-S-transferases (GSTs), and multidrug resistance-associated proteins (MRPs), underpinning its critical roles in hepatic toxicodynamics and toxicokinetics. Additionally, Nrf2 mediates myocardial cytoprotection against oxidative and electrophilic stress [4]. Unlike Nrf1/2, Nrf3 positively regulates redox signaling by promoting ROS generation rather than suppressing it. As an N-glycosylated protein located to the endoplasmic reticulum (ER), Nrf3 enhances ROS production and facilitated smooth muscle cell differentiation from stem cells [5]. In addition, Nrf3 could attenuate cell metastasis in triple-negative breast cancer cells (TNBC) by increasing cellular ROS accumulation and subsequently inhibiting ERK activation [6]. However, the roles of Nrf3 and its mediated ROS signaling in CMs death, I/R injury and pathological cardiac remodeling after MI remain poorly understood.

In an elegant study, Chen et al. [7] demonstrated that during MI, Nrf3 in CMs promotes mitochondrial ROS generation, thereby exacerbating cardiomyocyte apoptosis and adverse cardiac remodeling. Their findings reveal that Nrf3 exerts this detrimental effect by suppressing paired-like homeodomain transcription factor 2 (Pitx2) expression.

First, the researchers observed that Nrf3 is predominantly expressed in CMs of healthy hearts, while its expression is significantly upregulated in CMs from MI patients. This pattern suggests that Nrf3 has potential roles in both CM homeostasis and pathological processes involved in MI [7]. Under physiological conditions, Nrf3 is upregulated and translocated to the nucleus upon ER stress [5]. Given that cardiac injury can induce ER stress, Chen et al. [7] further demonstrated increased Nrf3 expression and nuclear accumulation in infarcted CMs and in cells exposed to an ER stress inducer or H₂O₂. Importantly, these effects were attenuated by an ER stress inhibitor, indicating that cardiac injury-induced ER stress contributes to Nrf3 activation and upregulation under pathological conditions. To further investigate the role of Nrf3 in MI, the researchers employed a series of genetically modified mouse models, including global Nrf3 knockout (Nrf3-KO), CM-specific knockout (Nrf3^△CM), and adeno-associated virus-mediated Nrf3-overexpressing (AAV9-Nrf3) mice. When subjected to MI or I/R models, these mice studies demonstrated that both global Nrf3 deficiency and cardiac-specific Nrf3 deficiency significantly reduced mortality, ameliorated adverse cardiac remodeling, and improved cardiac functions induced by MI or I/R injury [7].

Previous studies have reported that Nrf3 promotes ROS generation during stem cell differentiation [5]. Given the well-established role of mitochondrial ROS in the pathogenesis of MI [1], it is plausible to hypothesize that Nrf3-mediated ROS production may contribute to post-MI pathological phenotypes. Indeed, both MitoSOX and dihydroethidium (DHE) staining analyses showed that Nrf3^△CM mice exhibited significantly reduced mitochondrial ROS levels and markedly decreased MI-induced CMs apoptosis. Furthermore, using the mitochondria-targeted antioxidant MitoT and pro-oxidant MitoPQ, the researchers established that cardiac-specific Nrf3 knockout attenuates CMs apoptosis, reduces cardiac fibrosis, and improves cardiac function, all through the specific regulation of mitochondrial ROS production in both MI and I/R mouse models [7].

Pitx2 is a critical regulator of cardiogenesis with pleiotropic roles in cardiac development and disease. Deficiency in Pitx2 increases susceptibility to atrial fibrillation (AF) and atrial arrhythmia, as well as compromising neonatal regenerative capacity. Given that oxidative stress causes mitochondrial and electrophysiological dysfunction to promote AF and proarrhythmic remodeling in Pitx2 deficiency [8], Tao et al. [9] demonstrated that Pitx2 promotes cardiac repair by activating an antioxidant response through the upregulation of electron transport chain (ETC) components and ROS-scavenging enzymes. Furthermore, Pitx2 deficiency induces mitochondrial dysfunction and a metabolic change in human atrial cardiomyocytes [10]. Li et al. [11] reported that Pitx2 maintains mitochondrial integrity and function during cardiac regeneration, thereby preventing pathological myocardial fat deposition.

The study demonstrated that Pitx2 acts as a direct transcriptional target of Nrf3 in the context of cardiac injury. This is supported by findings that Nrf3 overexpression significantly suppresses Pitx2 expression, whereas CM-specific Nrf3 deletion markedly upregulates Pitx2 in both CMs and infarcted hearts. Strikingly, cardiomyocyte-specific knockdown of Pitx2 attenuates the protective effect of Nrf3 deficiency on cardiac function and remodeling following MI in Nrf3^△CM mice. Conversely, overexpression of Pitx2 significantly improves cardiac function, reduces mitochondrial ROS generation, and prevents cardiomyocyte apoptosis. Since the promoter region of Pitx2 contains cytosine-phosphate-guanine (CpG) islands, epigenetic modification by hypermethylation may reduce Pitx2 expression in CMs. Kao et al. [12] reported that heart failure induces Pitx2 promotor methylation via DNA methyltransferase (DNMT1) and then decreased Pitx2 levels in CMs. With multiple approaches, Chen et al. [7] demonstrated that Nrf3 recruits the hnRNPK-DNMT1 complex to induce methylation of the Pitx2 gene promoter, thereby suppressing its expression, which was counteracted by DNMT1-specific siRNAs (Fig. 1). Given that DNMT inhibitors have shown considerable promise in cancer therapy via epigenetic reprogramming, future research should translate these insights into novel therapeutic strategies in cardiac diseases pathogenesis.

Strikingly, Nrf2, a homolog of Nrf3, exerts opposite effects in CMs. Nrf2 not only directly activates Pitx2 expression, but also binds to cytoplasmic Pitx2 and shuttles it to nuclei, thereby activating the antioxidant response after cardiac injury [9].This indicates that Nrf3 and Nrf2 play antagonistic roles via the competitive regulation of Pitx2 expression during injury-induced cardiac remodeling. Pathologically, Nrf3 deficiency exacerbates CMs apoptosis and cardiac dysfunction, whereas Nrf2 confers cardio protection by coordinating antioxidant, anti-inflammatory, and metabolic reprogramming pathways. Functioning as positive and negative regulators of antioxidant response element (ARE)-mediated gene expression, respectively, Nrf2 and Nrf3 fine-tune ROS balance in tissue homeostasis and disease pathogenesis. Collectively, these findings clearly establish that Pitx2 activation or upregulation in CMs serves as a critical countermeasure against MI-induced CM apoptosis, cardiac dysfunction, and pathological remodeling, effects that closely resemble the cardioprotective phenotype observed upon Nrf3 deletion.

Mitochondrial ROS, a natural byproduct of aerobic metabolism, exists at a relatively stable concentration within the body. Low concentration of ROS acts as an “oxidation-reduction messenger,” facilitating intracellular signal transduction and regulation. ROS plays critical roles in the physiological activity, including sustaining the cell cycle, regulating gene expression, promoting cell proliferation, migration, and differentiation, as well as defending against environmental pathogens. Nrf3, which is expressed in CMs in healthy hearts, has been shown to bind to ARE and suppress the expression of the antioxidant gene Pitx2 [7]. The negative regulation is required because the low concentration of ROS is necessary to keep the cellular redox homeostasis. However, under pathological conditions, high concentration of ROS have deleterious effects, including causing severe oxidative damage to proteins, lipids, and DNA. Mitochondrial DNA is particularly sensitive to ROS-induced damage, which exacerbates mitochondrial dysfunction and activates mitochondria-mediated apoptosis of CMs, leading to a vicious cycle [13]. It is well established that ischemic preconditioning (IPC), initially described by Murry et al. [14], is a potent cardioprotective strategy against I/R injury. Notably, low levels of ROS can serve as a trigger for IPC, functioning as signaling molecules that protect against oxidative stress initiated by prolonged ischemia and successive reperfusion [15]. Consequently, precise regulation of cellular redox balance is important for both the prevention and treatment of MI.

Acute MI typically results from coronary artery occlusion due to atherosclerotic plaque rupture. Clinically, timely reperfusion using percutaneous coronary intervention (PCI) can effectively limit ischemic damage by restoring blood flow after MI [16]. However, reperfusion paradoxically triggers a burst of ROS production, causing significant CMs injury and adverse ventricular remodelling. While preclinical studies demonstrate promising results for antioxidant and anti-inflammatory therapies administered at reperfusion onset [16], their translation to human patients has proven challenging. This discrepancy stems not only from anatomical and physiological differences between animal models and humans, but also from the fact that young rodent models commonly used in research possess robust endogenous repair capabilities and well-maintained immune homeostasis, which markedly contrasts with the aging-associated pathophysiology of human heart failure patients. Notably, Chen et al. [7] identified elevated Nrf3 expression in infarcted human myocardium, and further demonstrated that Nrf3 promotes mitochondrial ROS generation and apoptosis in human induced pluripotent stem cell (iPSC)-derived CMs under oxidative stress [7], highlighting its significant potential for clinical translation.

In summary, Chen et al. [7] have identified Nrf3 as a novel and critical regulator of mitochondrial ROS production and CM apoptosis in MI. They first elucidated a critical pathophysiological pathway underlying myocardial injury and highlighted a previously unrecognized Nrf3-Pitx2 regulatory axis. Their findings establish Nrf3-Pitx2 signaling as a promising therapeutic target with significant potential for clinical translation in developing next-generation cardioprotective therapies.

References

[1] Bugger H , Pfeil K . Mitochondrial ROS in myocardial ischemia reperfusion and remodeling. Biochim Biophys Acta Mol Basis Dis 2020; 1866(7): 165768

[2] Kobayashi A , Ito E , Toki T , Kogame K , Takahashi S , Igarashi K , Hayashi N , Yamamoto M . Molecular cloning and functional characterization of a new Cap'n' collar family transcription factor Nrf3. J Biol Chem 1999; 274(10): 6443–6452

[3] Zhang Y , Chen X , Wang M , Zhu Y , Shi W , Li C , Zhang Z , Taniguchi H , Ao P . Nrf1 acts as a highly-conserved determinon for maintaining robust redox homeostasis in the eco-evo-devo process of life histories. Cell Stress 2025; 9: 65–142

[4] Fiori E , Davinelli S , Ferrera A , Medoro A , Barsali C , Battistoni A , Volterrani M , Volpe M , Saso L , Rubattu S . The emerging role of Nrf2 in heart failure: from cardioprotection to therapeutic approaches. ESC Heart Fail 2025; 12(6): 4000–4020

[5] Xiao Q , Pepe AE , Wang G , Luo Z , Zhang L , Zeng L , Zhang Z , Hu Y , Ye S , Xu Q . Nrf3-Pla2g7 interaction plays an essential role in smooth muscle differentiation from stem cells. Arterioscler Thromb Vasc Biol 2012; 32(3): 730–744

[6] Zheng C , Pan Y , Lin B , Li J , Chen Q , Zheng Z . NRF3 suppresses the metastasis of triple-negative breast cancer cells by inhibiting ERK activation in a ROS-dependent manner. Histol Histopathol 2025; 40(1): 123–131

[7] Chen Q , Zheng A , Xu X , Shi Z , Yang M , Sun S , Wang L , Wang Y , Zhao H , Xiao Q , Zhang L . Nrf3-mediated mitochondrial superoxide promotes cardiomyocyte apoptosis and impairs cardiac functions by suppressing Pitx2. Circulation 2025; 151(14): 1024–1046

[8] Subati T , Kim K , Yang Z , Murphy MB , Van Amburg JC , Christopher IL , Dougherty OP , Woodall KK , Smart CD , Johnson JE , Fogo AB , Amarnath V , Agrawal V , Barnett JV , Saffitz JE , Murray KT . Oxidative stress causes mitochondrial and electrophysiologic dysfunction to promote atrial fibrillation in Pitx2^+/− mice. Circ Arrhythm Electrophysiol 2025; 18(3): e013199

[9] Tao G , Kahr PC , Morikawa Y , Zhang M , Rahmani M , Heallen TR , Li L , Sun Z , Olson EN , Amendt BA , Martin JF . Pitx2 promotes heart repair by activating the antioxidant response after cardiac injury. Nature 2016; 534(7605): 119–123

[10] Reyat JS , Sommerfeld LC , O’Reilly M , Roth Cardoso V , Thiemann E , Khan AO , O’Shea C , Harder S , Müller C , Barlow J , Stapley RJ , Chua W , Kabir SN , Grech O , Hummel O , Hübner N , Kääb S , Mont L , Hatem SN , Winters J , Zeemering S , Morgan NV , Rayes J , Gehmlich K , Stoll M , Brand T , Schweizer M , Piasecki A , Schotten U , Gkoutos GV , Lorenz K , Cuello F , Kirchhof P , Fabritz L . PITX2 deficiency leads to atrial mitochondrial dysfunction. Cardiovasc Res 2024; 120(15): 1907–1923

[11] Li L , Tao G , Hill MC , Zhang M , Morikawa Y , Martin JF . Pitx2 maintains mitochondrial function during regeneration to prevent myocardial fat deposition. Development 2018; 145(18): dev168609

[12] Kao YH , Chen YC , Chung CC , Lien GS , Chen SA , Kuo CC , Chen YJ . Heart failure and angiotensin II modulate atrial Pitx2c promotor methylation. Clin Exp Pharmacol Physiol 2013; 40(6): 379–384

[13] Dubois-Deruy E , Peugnet V , Turkieh A , Pinet F . Oxidative stress in cardiovascular diseases. Antioxidants 2020; 9(9): 864

[14] Murry CE , Jennings RB , Reimer KA . Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986; 74(5): 1124–1136

[15] Hausenloy DJ , Barrabes JA , Bøtker HE , Davidson SM , Di Lisa F , Downey J , Engstrom T , Ferdinandy P , Carbrera-Fuentes HA , Heusch G , Ibanez B , Iliodromitis EK , Inserte J , Jennings R , Kalia N , Kharbanda R , Lecour S , Marber M , Miura T , Ovize M , Perez-Pinzon MA , Piper HM , Przyklenk K , Schmidt MR , Redington A , Ruiz-Meana M , Vilahur G , Vinten-Johansen J , Yellon DM , Garcia-Dorado D . Ischaemic conditioning and targeting reperfusion injury: a 30 year voyage of discovery. Basic Res Cardiol 2016; 111(6): 70

[16] Bhatt DL , Lopes RD , Harrington RA . Diagnosis and treatment of acute coronary syndromes: a review. JAMA 2022; 327(7): 662–675

RIGHTS & PERMISSIONS

Higher Education Press