Acute myocardial infarction (AMI), an ischemic heart disease caused by coronary artery blockage, reduces blood flow to the heart, resulting in high mortality/morbidity. Reperfusion therapy, though standard treatment, paradoxically worsens MI/RI. MI/RI involves calcium overload, inflammatory cytokines, mitochondrial dysfunction, autophagy, apoptosis, and ferroptosis [1]. Moreover, endothelial dysfunction is a critical determinant in the pathological cascade of myocardial infarction [2]. CMECs, the predominant cellular component of the myocardium, outnumber cardiomyocytes approximately twofold [3] and exhibit high susceptibility to H/R-induced injury. Despite these insights, numerous molecular mechanisms linking cardiac microvascular damage to MI/RI remain uncharacterized.

Mitochondria regulate metabolism (TCA cycle, oxidative phosphorylation) and cell signaling (Ca²⁺, immunity, cell death). Their dysfunction contributes to heart diseases like ischemia reperfusion (I/R) injury and heart failure [4]. Dysfunctional mitochondria exacerbate reactive oxygen species (ROS) production due to impaired electron transport chains, thereby intensifying oxidative stress and diminishing energy production in cardiomyocytes [[5], [6], [7]]. Mitochondrial dysfunction has been extensively demonstrated as a pivotal mechanism underlying cardiac microvascular impairment induced by I/R injury [8,9]. Damaged mitochondria produce excess superoxide, which oxidizes BH4 to BH2. This disrupts BH4-eNOS binding, causing eNOS dysfunction and reduced nitric oxide production [10]. Furthermore, mitochondrial-derived pro-apoptotic factors, including cytochrome c and Smac, translocate into the cytosol where they initiate caspase cascade activation, ultimately triggering endothelial cell apoptosis [11]. Beyond these well-characterized roles, emerging evidence highlights mitochondria as central regulators of multiple endothelial cell functions, encompassing mobilization, senescence, growth and proliferative capacity [12,13]. It is now recognized that mitochondrial structural disorder precedes mitochondrial dysfunction [14]. Increased mitochondrial fission reduces the mitochondrial membrane potential (MMP), augments mitochondrial reactive oxygen species (mtROS) production and activates mitochondria-dependent apoptotic pathways during cardiac microvascular I/R injury [15]. Consequently, therapeutic approaches targeting the restoration of CMECs mitochondrial integrity and function present a promising avenue for mitigating the progression of MI/RI.

Mitochondrial complex I is the largest enzyme in the respiratory chain, consisting of 45 subunits (14 core and 31 accessory). As the entry point of the respiratory chain, it catalyzes electron transfer from NADH to coenzyme Q10 while pumping protons across the mitochondrial inner membrane [16]. This proton translocation across the inner mitochondrial membrane generates an electrochemical gradient, which drives ATP synthesis in complex V. Simultaneously, ROS are produced as by-products in several mitochondrial complexes, predominantly in complex I [17]. Among the core subunits, NDUFS1, encoded by a nuclear gene, is the largest and is integral to the eight iron-sulfur clusters responsible for NADH oxidation [18,19]. As the initiation point of the mitochondrial electron transport chain (ETC), NDUFS1 plays a central role in metabolic reprogramming, oxidative stress, and apoptosis across various pathological conditions [20,21]. Despite its significance, the precise role of NDUFS1 in the pathophysiology of MI/RI remains poorly understood.

Nar (molecular formula: C27H32O14; molecular weight: 580.54 g/mol), a flavonoid derived from citrus plants and Chinese herbs, exhibits diverse biological activities, including antihypertensive, antioxidant, and anti-inflammatory effects [22]. Extensive research has highlighted its protective effects against oxidative stress [23], inflammatory responses [24], metabolic syndrome [25], neurological disorders [26], cardiovascular dysfunction [27], and respiratory diseases [28]. In recent years, Nar has been increasingly incorporated into dietary supplement formulations as a phytopharmaceutical agent [29]. In addition, Nar has been shown to inhibit fructose-induced myocardial hypertrophy [30] and normalize systolic blood pressure in high-fat diet-fed mice [31].

In this study, we demonstrated that Nar exerts a potent inhibitory effect on ferroptosis in myocardial tissue following MI/RI. More importantly, we have uncovered a novel molecular mechanism by which Nar exerts its cardioprotective effects: it specifically enhances IRF3 phosphorylation, thereby activating the crucial SLC7A11/Gpx4 antioxidant pathway both in vivo and in CMECs. Furthermore, our research revealed that Nar directly interacts with NDUFS1 in CMECs, promoting its mitochondrial translocation and subsequently improving mitochondrial function which is a previously unrecognized mechanism underlying Nar's therapeutic potential against MI/RI. These findings provided new insights into the multifaceted cardioprotective mechanisms of Nar, establishing it as a promising therapeutic agent for ischemic heart disease.