MiR-130a-3p Regulates FUNDC1-Mediated Mitophagy by Targeting GJA1 in Myocardial Ischemia/Reperfusion Injury

doi:10.21203/rs.3.rs-1740501/v2

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MiR-130a-3p Regulates FUNDC1-Mediated Mitophagy by Targeting GJA1 in Myocardial Ischemia/Reperfusion Injury

https://doi.org/10.21203/rs.3.rs-1740501/v2

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Understanding the complex pathogenesis in myocardial ischemia/reperfusion (I/R) injury (IRI) is an urgent problem in clinical trials. Increasing pieces of evidence have suggested that miRNAs are involved in the occurrence and development of heart diseases by regulating mitochondria-related gene expression. Mitochondria have been acknowledged as the key triggers of cardiac I/R injury. However, the potential impact of miR-130a on mitochondria remains unclear in myocardial IRI. Exploring the regulatory mechanism of miR-130a on mitochondria may provide a new target for IRI therapy. In the present study, we found that miR-130a significantly increased in acute myocardial infarction (AMI) patients and myocardial I/R rats. MiR-130a could downregulate the viability of cardiomyocytes and the knockdown of miR-130a could protect the viability of cardiomyocytes under hypoxia-reoxygenation (HR). Over-expression of miR-130a resulted in mitochondrial dysfunction. It was evidenced by decreases in mitochondrial ATP production, mitochondrial membrane potential (MMP), and an increase in reactive oxygen species (ROS) production. However, suppression of miR-130a could protect against mitochondrial damage, show elevation of mitochondrial ATP production rate and MMP, and reduce ROS production. We further explored the effect of miR-130a on the mitochondrial quality control (QMC) system by determining mitochondrial-protein-specific proteases and analyzed mitochondrial morphology by fluorescence imaging and electron microscopy, respectively. It was noted that miR-130a could suppress mitochondrial fusion and FUNDC1-mediated mitophagy to accelerate myocardial IRI. Moreover, we investigated the potential miR-130a targeted mitochondria-related genes to understand the regulatory mechanism of miR-130a in the setting of myocardial IRI. It was revealed that miR-130a targeted GJA1, and GJA1 rescued IRI by enhancing ATP production rate and oxidative phosphorylation, meanwhile protecting cell viability, MMP, and activating mitophagy. In addition, the knockdown of miR-130a significantly activated FUNDC1-mediated mitophagy, while the knockdown of GJA1 reversed the relevant response. Collectively, our findings suggest that miR-130a regulates FUNDC1-mediated mitophagy by targeting GJA1 in myocardial IRI.

miR-130a

Myocardial ischemia/reperfusion injury

GJA1

Mitophagy

Mitochondria

Myocardial Ischemia/reperfusion (I/R) injury is a common and serious pathophysiological phenomenon during cardiac surgery, heart transplant, MI, and cardiac arrest (1). Due to its complex pathogenesis, there is still no effective method to treat myocardial I/R injury (IRI) in clinical practice (2-5). Understanding the complex pathogenesis of myocardial IRI is an urgent problem solved in clinical trials (1). MiRNAs and mitochondria are critical to involve in cardiac pathophysiology (6). miRNAs play an important role in the pathogenesis and prognosis of cardiac diseases, and can also be an important therapeutic target (7). MiRNAs are a class of short and highly conserved non-coding RNAs (8). Increasing pieces of evidence have suggested that miRNAs are involved in the occurrence and development of heart disease by regulating the mitochondria-related genes, including biogenesis, fission, and mitophagy genes (9, 10). MiR-130a is a high-expression gene in the heart (11, 12). Current studies have reported that miR-130a is an important regulator involved in various cardiovascular diseases (11-15). However, the impact of miR-130a on mitochondrial function remains unknown. Mitochondria have been acknowledged as the key triggers of cardiac IRI (16). Mitochondria act as "energy factories", providing energy to all cells (17). As the most metabolically active organ, the heart needs a lot of mitochondria to provide energy for keeping systolic and diastolic functions. Mitochondrial dysfunction is associated with cardiac disease development and is caused by reduced ATP synthesis, excessive reactive oxygen species (ROS) production, the disorder of mitochondrial dynamics, and the abnormality of mitophagy (18). Emerging studies have suggested that mitochondrial homeostasis plays an important role in mitochondrial dysfunction, and mitophagy is a crucial process in regulating mitochondrial homeostasis (7). Exploring the regulation mechanism of miR-130a on mitochondria may provide a new target for myocardial IRI therapy.

MiRNAs regulate gene expression by inhibiting translation or inducing mRNA degradation (19). The genes targeted by miR-130a were predicted by the miRBase database, and GJA1 was chosen as the target for our study. Protein GJA1/CX43 is a major gap junction component in many cardiac biologic processes. Most of the previous studies on GJA1 focused on its role in information exchange and material transport between cells by forming gap junctions and semi-channels. However, studies have clarified GJA1 can regulate autophagy (20, 21) and hold mitochondrial protection in recent years (22, 23). GJA1 is a high-expression protein in the mitochondria of cardiomyocytes (24), and some studies have indicated that GJA1 is involved in the pathogenesis of myocardial IRI (25, 26). Nevertheless, the regulatory mechanism of GJA1 in myocardial IRI remains unclear.

In this study, we found miR-130a significantly increased in both acute myocardial infarction (AMI) patients and myocardial I/R rats. Mitochondria functional studies suggested that Overexpression of miR-130a could cause mitochondrial dysfunction, and suppression of miR-130a could protect mitochondria. Moreover, miR-130a suppressed mitochondrial fusion and FUNDC1-mediated mitophagy to accelerate myocardial HRI. Next, we found miR-130a targeted GJA1. And, GJA1 rescued IRI by enhancing ATP production rate and oxidative phosphorylation, meanwhile protecting cell viability, mitochondrial membrane potential, and activated mitophagy. At last, we confirmed knockdown of miR-130a could activate FUNDC1-mediated mitophagy, however, the knockdown of GJA1 could reverse the effect. Our findings demonstrated miR-130a regulated FUNDC1-mediated mitophagy by targeting GJA1 in myocardial IRI.

MiR-130a increased in AMI patients and I/R rats

To explore the role of miR-130a on myocardial IRI, we tested the miR-130a-3p expression in the peripheral blood of AMI patients treated with reperfusion. Characteristics of patients were shown in Fig. 2A. The level of miR-130a significantly increased in the AMI patients compared with the healthy person, as indicated in Fig. 2B. Next, the models of myocardial I/R rats were constructed, and Standard II lead electrocardiogram was observed during the experiment. As shown in Fig. 2C, the ST segment was elevated after myocardial ischemia and recovered after reperfusion. To investigate whether did cardiac function change in the I/R rats, cardiac parameters were measured by small animal ultrasound machines (FUJIFILM, Bothell, WA). Compared with the Sham group, HR, LVEF, FS, and LVPW; s significantly decreased in the I/R group (Fig. 2D-F). In addition, the heart histology and morphology were observed with HE staining. As shown in Fig. 2G, myocardial fibers were broken, and cardiomyocytes revealed slighter degeneration and necrosis in the I/R group. To further confirm the result that miR-130a increased in myocardial IRI, we performed an RT-qPCR test in the myocardial tissue of I/R rats and found the miR-130a expression in the I/R group was higher than that in the sham group (Fig. 2H). The above data indicated that miR-130a increased in myocardial IRI, and might play a vital role.

MiR-130a affected the viability of HR Cardiomyocytes

In the following, to explore the role of miR-130a on the viability of cardiomyocytes, we tested the activity and apoptosis of H9c2 cells under normoxia or HR conditions. As shown in Fig. 1, the over-expression of miR-130a could decrease the relative cell activity (Fig. 1D) and increase the apoptosis of H9c2 cells (Fig. 1B-C) under normoxia and HR conditions. Besides, suppression of miR-130a could increase the relative cell activity (Fig. 1D) and decrease the apoptosis of H9c2 cells (Fig. 1B-C) under normoxia and HR conditions. These results indicated that miR-130a could downregulate the viability of cardiomyocytes and the knockdown of miR-130a could protect the viability of cardiomyocytes.

MiR-130a affected mitochondrial function by regulating ATP production rate, oxidative phosphorylation, reactive oxygen species content, and mitochondrial membrane potential

To further confirm whether miR-130a played a vital role in myocardial IRI by targeting mitochondria, we examined ATP production rate, mitochondrial respiration, ROS content, and MMP in miR-130a over-expression (OE) and knockdown (KD) H9c2 cells during hypoxia-reoxygenation (HR). As shown in Fig. 3, the Over-expression of miR-130a could cause mitochondrial dysfunction in both the control and HR group, according to the decrease of ATP production rate (Fig. 3A-F), mitochondrial respiration (Fig. 3G-K), MMP (Fig. 3M&O), and the increase of ROS production (Fig. 3L&N). Besides, suppression of miR-130a could protect mitochondria in both the Ctrl (normoxia) and HR group, according to the increase of mitochondrial ATP production rate (Fig. 3A-F), mitochondrial respiration (Fig. 3G-K), MMP (Fig. 3M&O), and the decrease of ROS production (Fig. 3L&N). Taken together, these results indicated that the increase of miR-130a expression aggravated mitochondrial dysfunction in myocardial hypoxia-reoxygenation injury (HRI).

MiR-130a exerted a mitochondrial quality control system by mediating mitochondrial dynamics and mitophagy

The next question was, after mitochondrial dysfunction in myocardial IRI, how did the mechanisms responsible for the elimination of dysfunctional mitochondria work? Studies have shown mitochondrial quality control (QMC) system including mitochondrial dynamics and mitophagy, is essential for mitochondrial function following injury (27). Therefore, we explored whether miR-130a could further affect mitochondrial dynamics and mitophagy in myocardial IRI. There are some mitochondrial-protein-specific proteases in the QMC as follows: Mitofusins 1 and 2 (MFN1, MFN2) involve in the fusion, the GTP-dependent dynamin-related protein 1 (DRP1) regulates the fission, and Bcl2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), FUN14 domain containing 1 (FUNDC1), PARKIN, microtubule-associated protein 1A/1B-light chain 3 (LC3) cause mitophagy. Western blot (WB) results showed MFN1, MFN2, FUNDC1, and LC3Ⅱ expression significantly decreased in the HR NC group, compared with the Ctrl NC group. Similarly, miR-130a over-expression caused MFN1, MFN2, FUNDC1, and LC3Ⅱ expression to decrease under normoxia and HR conditions. Meanwhile, MFN1, FUNDC1, PINK1, and LC3Ⅱ expression significantly increased in the miR-130a KD group, compared with the NC group under normoxia and HR conditions (Fig. 4A-B). These results suggested that under HR condition mitochondrial fusion and FUNDC1-mediated mitophagy would be suppressed, and over-expression of miR-130a could increase the suppression effect. Moreover, suppression of miR-130a could promote mitochondrial fusion and FUNDC1-mediated mitophagy, which reversed the myocardial HRI.

A shift in the mitochondrial dynamics balance leads to changes in mitochondrial structural features (28). To further confirm miR-130a could affect MQC, we analyzed mitochondrial morphology by fluorescence imaging and electron microscopy (EM). As shown in Fig. 4C&E&D, small fragmentary mitochondria would generate under HR condition and mitochondrial swelling. Similarly, over-expression of miR-130a would generate small fragmentary mitochondria and mitochondrial swelling. Meanwhile, suppression of miR-130a would generate a network of long tubular mitochondria (Fig. 4C&E) which are beneficial for cell metabolism. Mitophagy will be activated as the number of damaged mitochondria increases to protect myocardial cells (29). As shown in Fig. 4D&F, mitophagy was suppressed under the HR condition. Interestingly, mitophagy was suppressed in the miR-130a OE group and was activated in the miR-130a KD group (Fig. 4D&F). Taken together, these results stated above suggested the increase of miR-130a generated damaged mitochondria, and at the same time suppressed mitophagy to accelerate myocardial HRI, and suppression of miR-130a could rescue myocardial HRI.

GJA1 is the target of miR-130a and is down-regulated in I/R rats

We have demonstrated the increase of miR-130a aggravated mitochondrial dysfunction in myocardial IRI by suppressing mitophagy. To understand the injury mechanism of miR-130a, we first explored which mitochondria-related genes did miR-130a target. MiRDB database was used to predict the genes miR-130a-3p targeted. As shown in Fig. 5A, we noticed the target score of GJA1 was the highest among 922 predicted target genes. To verify whether miR-130a may target the GJA1 3’UTR and regulate GJA1, GP-miRGLO-GJA1-3’-UTR-WT, and GP-miRGLO-GJA1-3’-UTR-Mut luciferase reporter plasmids were designed (Fig. 5F). Luciferase reporter activities were decreased by miR-130a mimics in the wild type (WT) group but not in the mutant (MUT) group, indicating that miR-130a target GJA1 directly (Fig. 5G). Moreover, GJA1 expression could be affected by miR-130a over-expression or knockdown (Fig. 5B), indicating the regulating correlation between miR-130a and GJA1 protein. Our previous studies showed that miR-130a increased in myocardial IRI, which made us wonder whether the level of GJA1 expression changed accordingly. We measured GJA1 expression in myocardial I/R rats by WB (Fig. 5C), immunohistochemistry (IHC) (Fig. 5D), and immunofluorescence assay (IFA) (Fig. 5E), and show consistent results that GJA1 in the I/R group significantly decreased. Therefore, we reasoned that miR-130a aggravated mitochondrial dysfunction in myocardial IRI by regulating GJA1.

GJA1 enhanced ATP production rate and oxidative phosphorylation, protected cell viability and mitochondrial membrane potential, and activated mitophagy.

Based on the previous results, we reasoned that miR-130a aggravated mitochondrial dysfunction in myocardial IRI by regulating GJA1. To test this assumption, initially, we detected the protection of GJA1 on the cell viability and mitochondria through apoptosis, CCK8, ATP production rate, mitochondrial respiration, MMP, and mitophagy. The over-expression of GJA1 could increase the relative cell activity (Fig. 6C) and decrease the apoptosis of H9c2 cells (Fig. 6A-B) under normoxia and HR conditions. The Over-expression of GJA1 could protect mitochondria in both the Ctrl (normoxia) and HR groups, according to the increase of mitochondrial ATP production rate (Fig. 6D), mitochondrial respiration (Fig. 6E), MMP (Fig. 6F-G). To establish the role of GJA1 on mitophagy, we performed WB to assess the levels of FUNDC1-mediated mitophagy-associated proteins. It was evident that HR injury decreased FUNDC1 and LC3Ⅱ, however, over-expression of GJA1 significantly increased FUNDC1 and LC3Ⅱ levels (Fig. 6J). Further, we performed co-localization experiments to observe mitophagy activity where we found a significant decrease in co-localization of lysosomal and mitochondrial structures under HRI (Fig. 6H-I). However, GJA1 over-expression rescued this defect. Hence, these results indicated that GJA1 could rescue the cells under HRI by enhancing ATP production rate and oxidative phosphorylation, protecting MMP, and activating mitophagy.

MiR-130a regulated mitophagy through GJA1

We demonstrated that both miR-130a and GJA1 could regulate FUNDC1-mediated mitophagy, and miR-130a targeted the mitochondria-related gene GJA1. We further wanted to understand the potential effect of miR-130a on the mitochondrial functions of GJA1. Hence, we silenced GJA1 in the miR-130a KD cells under normoxia and HR conditions. Firstly, the mRNA and protein levels of GJA1 expression were detected. Both mRNA and protein levels of GJA1 in the miR-130a KD group significantly increased, and the knockdown of GJA1 could reverse the impact of miR-130a knockdown on GJA1 levels (Fig. 7A-B) under normoxia and HR conditions. Secondly, to identify the association between miR-130a and GJA1 on mitophagy, we performed WB to assess the levels of FUNDC1-mediated mitophagy-associated proteins. As shown in Fig. 7A, the levels of LC3 II and FUNDC1 expression significantly increased in the miR-130a KD group, however, the knockdown of GJA1 in the miR-130a KD group could reverse the effect under Ctrl (normoxia) and HR conditions. Further, we performed co-localization experiments to observe mitophagy activity where we found a significant increase in co-localization of lysosomal and mitochondrial structures in the miR-130a KD group under Ctrl (normoxia) and HR conditions (Fig. 7C-D). However, the knockdown of GJA1 rescued this defect (Fig. 7C-D). Therefore, these results suggest that GJA1 was required for miR-130a to aggravate mitochondrial dysfunction in myocardial HRI by regulating FUNDC1-mediated mitophagy.

Myocardial IRI often occurs during reperfusion therapy of AMI (30). For patients with AMI, interventional therapy and coronary artery bypass grafting are the main clinical treatment methods (31). Reperfusion therapy can clear blocked coronary arteries and restore blood flow to the myocardium, nevertheless, it might result in myocardial IRI. Therefore, the diagnosis and treatment of myocardial IRI is an important way in the management of AMI. In the current study, we found the level of miR-130a was significantly increased in the AMI patients. Interestingly, the increase of miR-130a could suppress mitochondrial fusion and FUNDC1-mediated mitophagy to accelerate myocardial HRI. Furthermore, we identified that miR-130a regulated FUNDC1-mediated mitophagy to aggravate mitochondrial dysfunction by targeting GJA1.

Several studies have documented that miR-130a is highly expressed in the heart and plays an important role in the development of cardiovascular diseases (12, 13, 15, 32). MiR-130a has a protective effect on cardiovascular diseases, for instance, Feng et al. found that miR-130a attenuated cardiac fibrosis after myocardial infarction through transforming growth factor (TGF)-β/Smad signaling by directly targeting TGF-β receptor 1 (33). Also, there are some studies to find miR-130a might exert a damaging impact on rat cardiomyocytes, for example, down-regulation of miR-130 expression was noted to enhance cardiac protection through PPAR-γ by inhibiting inflammation and myocardial fibrosis (34). Due to its involvement in the regulation of multiple pathways, its complex mechanism of action remains controversial. Herein, we found miR-130a was significantly increased in the patients with AMI. Recent studies have demonstrated that miRNAs and mitochondria are critically involved in cardiac pathophysiology (35). Restoring and improving mitochondrial function in cardiomyocytes is an important therapeutic target for delaying the occurrence and development of cardiovascular diseases (36, 37). Mitochondria are the key triggers cause of cardiac I/R injury (16) because cardiomyocytes contain lots of mitochondria that supply more than 90% of the energy of cardiomyocytes (38) and can promote cardiomyocyte death by inducing apoptosis or necrosis after myocardial reperfusion. Therefore, we attempted to investigate whether the changes in miR-130a expression in hearts had an impact on mitochondrial function. In this study, we found that miR-130a KD could improve mitochondrial function, including an increase in ATP production rate and mitochondrial respiration, a decrease in ROS production, and rescue of MMP. On the contrary, we observed that miR-130a OE augmented mitochondrial dysfunction, including reduced ATP production rate and mitochondrial respiration, increased ROS production, and decreased MMP. As we know, mitochondria have evolved to generate a variety of quality control systems, which are multi-layered, acting on molecular, organelle, and cellular levels to ensure the normal structure and function of cell homeostasis as well as the mitochondrial network (39). Studies have shown QMC system including mitochondrial dynamics and mitophagy, is essential for mitochondrial function following injury (27). After mitochondrial dysfunction in myocardial IRI, how did the mechanisms responsible for the elimination of dysfunctional mitochondria work? To answer the question, we explored whether miR-130a could further affect mitochondrial dynamics and mitophagy. Our data indicated that miR-130a significantly reduced mitochondrial fusion, thereby leading to morphological fragmentation of mitochondria. Moreover, mitophagy appeared to be suppressed, which might contribute to the failure of fragmented mitochondria clearing. Mitophagy prevents the accumulation of abnormal mitochondria, while the suppression of mitophagy could lead to cardiomyocyte dysfunction or even death (40). Accumulating pieces of evidence have demonstrated that mitophagy is critically involved in the development of mitochondrial dysfunction in various settings (41). Of note, mitochondria can repair themselves through the mitophagy system, by delivering damaged mitochondrial fragments to lysosomes, to reduce the destruction caused by mitochondrial damage to cells, including reducing oxidative stress of cells and restoring mitochondrial energy metabolism (42). Herein, we further investigated the effect of miR-130a on the activation of the classical mitophagy pathway, in which only the expression of FUNDC1 was significantly altered. Over-expression and knockdown of miR-130a could respectively down-regulate and up-regulate the FUNDC1-mediated mitophagy. Taken together, we believe that there might be a novel signaling pathway involved in the process, that is, miR-130a directly participates in the modulation of FUNDC1-mediated mitophagy by targeting mitochondrial function-related genes.

To clarify the potential mechanism concerning miR-130a in mitophagy, initially, we predicted the target gene of miR-130a. Then, we chose GJA1 as the research object, for the highest score and the mitochondrial metabolic protection of GJA1 (43). And, GJA1 expression could be affected by miR-130a over-expression or knockdown, indicating the regulating correlation between miR-130a and GJA1. Emerging evidence has indicated that GJA1/CX43 is the main gap junction component. In addition to the formation of gap junctions and semi-channels, novel functions of GJA1 have been documented in recent years, such as mitochondrial function and autophagy modulation [15, 16, 31]. A study by Boengler et al. revealed that GJA1 played an important role in the preservation of mitochondrial function by regulating ROS content and ATP production (22). In this study, we further noticed that GJA1 could markedly enhance ATP production rate and oxidative phosphorylation, meanwhile could protect the viability and the MMP of rat cardiomyocytes in HRI. It was reported that GJA1 maintained cellular connectivity and homeostasis by regulating autophagy (44). Martins-Marques et al. found that GJA1 was degraded in the setting of I/R, and it was correlated to autophagic activation (26). Mitophagy is a form of macro autophagy. Given the regulatory role of GJA1 in autophagy and mitochondrial function, whether GJA1 could modulate mitophagy is worthy of further investigation. Our results that GJA1 could activate FUNDC1-mediated mitophagy might deepen the understanding of the mechanism underlying GJA1-associated mitophagy in cardiomyocytes.

Given the question of whether the regulation of mitophagy by miR-130a was implemented through GJA1, we found that the activation of FUNDC1-mediated mitophagy was markedly reversed after the knockdown of GJA1 in miR-130a KD cells. These results indicated that miR-130a suppressed the activation of FUNDC1-mediated mitophagy via GJA1. The mitochondrial-protein-specific proteases BNIP3, FUNDC1, and Parkin in the QMC are essential receptors in mediating the activity of mitophagy. FUNDC1 is the only upstream regulator found to moderately activate autophagy for mitochondrial protection. A recent report suggested that FUNDC1-induced mitophagy exerted a protective effect on IRI (45). Mitophagy receptor-FUNDC1 located in the mitochondrial outer membrane can bind to the interaction domain of the key autophagy protein LC3 to activate mitophagy and plays an important role in hypoxia-regulated mitophagy (46). Under normal conditions, FUNDC1 is phosphorylated by the tyrosine kinase and thus has a low affinity for LC3 (47). Upon hypoxia, the inactivation of tyrosine kinase increases affinity between FUNDC1 and LC3 and further induces mitophagy (47). In this study, GJA1 was noted to activate FUNDC1-mediated mitophagy. We reasoned that GJA1 might catalyze the binding of FUNDC1 to LC3 and activate mitophagy by augmenting FUNDC1 dephosphorylation. Therefore, our research on the regulation of FUNDC1-mediated mitophagy might fill the blank on the upstream regulatory signal of FUNDC1 phosphorylation in IRI.

This study must be interpreted in the context of certain limitations. First, we have only studied miR-130a regulated FUNDC1-mediated mitophagy to aggravate mitochondrial dysfunction by targeting GJA1 in vitro; however, further research is needed to verify this effect in vivo, which will be performed in our ongoing project. Second, the GJA1-FUNDC1 pathway is potentially signaling in mediating the dysfunction of cardiomyocytes, and its interaction network regarding mitophagy needs to be clarified. Third, we have studied the impact of miR-130a on myocardial IRI-related mitophagy and its underlying mechanism, nonetheless, further investigation is required to definite whether miR-130a has clinical significance in myocardial IRI.

In summary, our data suggest that miR-130a regulates the activation of FUNDC1-mediated mitophagy by targeting GJA1. MiR-130a involves in myocardial IRI by down-regulating GJA1 and suppressing FUNDC1-related mitophagy (Fig. 8). MiR-130a and the GJA1-FUNDC1-associated mitophagy pathway might be potential targets in new-drug development for myocardial IRI, thereby providing a novel therapeutic strategy to combat AMI.

Patient population

Blood samples of AMI patients were collected from the Cardiology Department of the Chinese PLA General Hospital (n = 4), Beijing, China. For our study, the diagnosis of AMI was made according to the inclusion criteria from the World Health Organization definition of myocardial infarction: 2008-2009 revision (48). Informed consent for participation in the study has been obtained, and this consent was written. The study was approved by the Ethical Committee for Medical Research of Chinese PLA General Hospital, Beijing, China.

Cell culture

The H9c2 rat cardiomyoblast cell line was obtained from the Qianzhao Xinye Biology Science and Technology Co., Ltd, Beijing, China. The H9c2 cells were recently authenticated by STR profiling and were tested free of mycoplasma contamination. The H9c2 cells were grown in complete medium with 10% fetal bovine serum (FBS) (Invitrogen, 10100147, Carlsbad, CA), 1% penicillin-streptomycin, and DMEM high sugar medium (Invitrogen, 11965092, Carlsbad, CA) under 20% O₂ and 5% CO₂ at 37 °C.

Rat myocardial I/R models construction and hypoxia-reoxygenation (HR) treatment of H9c2 cells

The animals and cell lines used in this study were fully compliant with the Guide for the Use of Laboratory Animals published by the US National Institutes of Health. The animal experiment procedure was approved by the Experimental Animal Ethics Committee of the Chinese PLA General Hospital, Beijing, China. Sprague-Dawley (SD) rats (male, 10 weeks, n = 12) were purchased from SPF (Beijing) Biotechnology Co., Ltd. The rats were narcotized with 1% Amytal sodium (6 ml/kg), and then were intubated and ventilated by the animal ventilator (Kent Scientific Corporation, S-MVG-RT, Torrington, WY). After a left thoracotomy, the proximal left anterior descending (LAD) coronary artery was ligated with a 5.0 Prolene suture to induce myocardial ischemia. Ischemia was performed for 45 minutes followed by reperfusion for 6 hours, and the heart samples were obtained for experiments. After reperfusion, cardiac parameters were measured by small animal color ultrasound (Fujifilm Visualsonics, Inc., Vevo3100, Bothell, WA) including left ventricular ejection fraction (LVEF), fractional shortening (FS), left ventricular posterior wall; diastolic (LVPW; d), left ventricular posterior wall; systolic (LVPW; s), and heart rate (HR). The sham group underwent the same procedures but did not ligate LAD after threading. The animal sample size was chosen by considering both the specimen availability and experience. In addition, no samples were excluded from the analysis and all rats were randomly assigned to the experiments. There was no blinding method used for the group allocation during experiments. To Simulate IRI in vitro, cells were cultured in Hanks' balanced salt solution (Invitrogen, 13150016, Carlsbad, CA) under 95% N₂ and 5% CO₂ for 45 minutes, then under normal oxygen conditions while the medium was changed to DMEM re-oxygenating for 6 hours.

Experimental protocol

The grouping and treatment of SD rats and H9c2 cells in the present study were shown in Fig. 1A.

Plasmids, siRNA, and inhibitor transfection

For miR-130a OE plasmid construction, it was subcloned rno-miR-130a-3p-sponges into CMV-EGFP-MCS-SV40-neomycin vector. For the knockdown of miR-130a, we used the miR-130a (rat) inhibitor (5'-AUGCCCUUUUAACAUUGCACUG-3'). For GJA1 OE plasmid construction, it was subcloned GJA1 (rat), accession no: NC_051355.1 into pRP[Exp]-NeoR-CMV vector. For the knockdown of GJA1, we used the siRNA_GJA1 sequences for GJA1 (rat) (Forward: 5'-GCCGCAAUUACAACAAGCATT-3'; Reverse: 5'-UGCUUGUUGUAAUUGCGGCTT-3'). The plasmids, siRNA, and inhibitor were transfected with Lipofectamine^TM 3000 Transfection Reagent (Invitrogen, L3000015, Carlsbad, CA), and the cells were collected for subsequent experiments after being cultured for 48 hours.

Databases

The genes targeted by hsa-miR-130a-3p were computationally predicted utilizing the miRDB database (http://www.mirdb.org/).

Measurement of gene expression by quantificational reverse transcription-polymerase chain reaction (qRT-PCR)

According to the instructions, total RNA was extracted using RNAqueous® Total RNA Isolation Kit (Thermo Fisher Scientific Inc, AM1912, Waltham, MA) and was reverse transcribed using PrimeScript^TM RT reagent Kit (Takara Biomedical Technology Co., Ltd., RR047A, Osaka, Japan) for GJA1 analysis. The mRNA level of the GJA1 gene was detected by qRT-PCR using GoTaq qPCR Master Mix Kit (Promega Corporation, A6001, Madison, WI), with β-Actin as a reference gene. For miR-130a analysis, total RNA was reverse transcribed with miRNA ALL-In-One cDNA Synthesis Kit (abm Inc., Zhenjiang, China), and the level of miR-130a was detected using GoTaq qPCR Master Mix Kit, with U6 as an internal control. MiR-130a (rat) specific primer and universal 3’ miRNA reverse primer were purchased from Applied Biological Materials Inc., Richmond, Canada. Other primers were as follows: GJA1 (rat) forward 5′-GCTGGTGGTGTCCTTGGT-3′, reverse 5′-AGTGGAGCCGTTGGTGAG-3′; U6 (rat) forward 5′-CCTGCTTCGGCAGCACA-3′, reverse 5′-AACGCTTCACGAATTTGCGT-3′; β-Actin (rat) forward 5’-CCAACCGTGAAAAGATGACC-3’, reverse 5’-ACCAGAGGCATACAGGGACA-3’.

Western blot

H9c2 cells and rat myocardial tissues were lysed with RIPA lysis buffer containing PMSF (Coolaber, SL1020, Beijing, China) for protein extraction. Protein was separated by 10% SDS-PAGE (Bio-Rad Laboratories, Inc., 5671034, Hercules, CA) and transferred to PVDF transfer membranes (Thermo Fisher Scientific Inc, 88520, Waltham, MA). After blocking, the PVDF membranes were incubated overnight with primary antibodies at 4°C. Then, the PVDF membranes were incubated with a secondary antibody at room temperature for 2 hours (1:3000, Abcam, ab6721, Cambridge, England). The images were obtained by the detector (General Electric Company, Imager 600 Amersham Imager 600 series, Boston, MA) using chemiluminescence detection reagents (Applygen Technologies Inc., Beijing, China). Primary antibodies against FUNDC1 (Cell Signaling Technology, 49240, Boston, MA), Parkin (Cell Signaling Technology, 4211, Boston, MA), BNIP3 (Cell Signaling Technology, 12396, Boston, MA), LC3A/B (Cell Signaling Technology, 4108, Boston, MA), GJA1 (Cell Signaling Technology, 83649, Boston, MA), MFN1 (Proteintech Group, Inc., 13798-1-AP, Chicago, IL), MFN2 (Proteintech Group, Inc., 12186-1-AP, Chicago, IL), DRP1 (Abcam, ab184247, Cambridge, England), and β-Actin (Cell Signaling Technology, 4970, Boston, MA) were used in this study, respectively.

Hematoxylin-eosin (HE) staining and immunohistochemistry (IHC) assay

Myocardial tissues of rats were in sequence dehydrated in gradient, embedded in paraffin, and cut into 5-micrometer slices. After dewaxing, the slices were stained by HE. For the IHC assay, the paraffin slices were dewaxed and treated with 3% hydrogen peroxide at room temperature for 15 minutes. The slices were boiled in EDTA solution for 3 minutes and then incubated overnight with GJA1 (Cell Signaling Technology, 83649, Boston, MA) antibody at 4°C after blocking. The slices were incubated with the second antibody at 37°C for 20 minutes, then stained the nucleus. The images were obtained under a microscope (Nikon Instruments Co., Ltd., Ts2, Shanghai, China). Optical density was measured by ImageJ software.

Immunofluorescence

Myocardial tissues of rats were in sequence dehydrated in gradient, embedded in paraffin, and cut into 5-micrometer slices. For the Immunofluorescence assay, the paraffin slices were dewaxed and treated with 3% hydrogen peroxide at room temperature for 30 minutes. The slices were blocked with 3% albumin from bovine serum (BSA) for 30 minutes. Then incubated overnight with GJA1 (1:500, Cell Signaling Technology, 83649, Boston, MA) antibody at 4°C after blocking. The slices were incubated with the second antibody (1:200, Abcam, ab150083, Cambridge, England) at 37°C for 30 minutes, then stained the nucleus. The images were obtained under a microscope (Nikon Instruments Co., Ltd., Ts2, Shanghai, China). Fluorescence intensity was measured by ImageJ software.

Luciferase Assay

GP-miRGLO-GJA1-3’-UTR-WT and GP-miRGLO-GJA1-3’-UTR-Mut luciferase reporter plasmids were obtained from GenePharma (Shanghai, China). 293T cells were planted in a 6-well cell culture plate (7.5×10⁴/well), and after 24h were co-transfected with luciferase reporter plasmids (2 μg) and miR-130a mimics (2 μg) with Lipofectamine^TM 3000 Transfection Reagent (Invitrogen, L3000015, Carlsbad, CA). Luciferase assay was performed using the Dual-Luciferase Reporter System (Promega, E1910, Madison, WI). Firefly and Renilla signals were measured using a chemiluminescent enzyme-labeled instrument (Molecular Devices, SpectraMax L, Sunnyvale, CA), and Firefly/Renillia were used to analyze luciferase activity.

Seahorse real-time ATP rate assay

The Agilent Seahorse XF Real-time ATP Rate Assay (Agilent Technologies, Inc., 103592-100, Santa Clara, CA) was designed to measure living cells’ total ATP production rate. After serial addition of mitochondrial inhibitors (oligomycin and rotenone/antimycin A, Rote/AA), Seahorse Analyzers directly measure real-time extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of cells-indicators of the two major energy-producing pathways: glycolysis and oxidative phosphorylation. After analysis, cells of each well were counted, and the results were normalized to the value per 5000 cells. The calculation of the mitochondrial and glycolytic ATP production rates provides a real-time measurement of cellular ATP production rates and a quantitative phenotype of cellular energy poise.

Seahorse mito stress assay

The Agilent Seahorse Cell Mito Stress Assay (Agilent Technologies, Inc., 103015-100, Santa Clara, CA) measures oxidative phosphorylation by directly measuring the OCR of cells. The compounds (oligomycin, FCCP, and a mix of Rote/AA) were serially injected to measure ATP production, maximal respiration, and nonmitochondrial respiration, respectively. Proton leak and spare respiratory capacity are then calculated using these parameters and basal respiration. After analysis, cells of each well were counted, and the results were normalized to the value per 5000 cells.

Apoptosis detection for live cells

Apoptosis detection for H9c2 cells was measured using flow cytometry (SONY, SH800, Tokyo, Japan). 10⁵ Cells/group were harvested and stained with Caspase-3 substrate (green fluorescence, Ex/Em=500/530nm) and Annexin V-mCherry (red fluorescence, Ex/Em=587/610nm) (Beyotime Biotechnology, C1077S, Shanghai, China) for 30 min at 25 ℃. Then flow cytometry was performed for apoptosis detection.

Cell counting kit 8 (CCK8) cell viability assay

The procedure was performed according to the instructions of the Cell Counting Kit-8 (Beyotime Biotechnology, C0038, Shanghai, China), which is widely used for the detection of cell proliferation and cytotoxicity. Briefly, H9c2 cells were seeded in 96-well plates at a concentration of 5×10⁴ per well and were incubated for 48 h. 10 μL of CCK-8 reagent was added to each well, and the absorbance was measured at 450nm after 2 h of incubation.

Detection of mitochondrial morphology by laser confocal microscopy

Cells were incubated with mito-tracker red (Beyotime Biotechnology, C1035, Shanghai, China) at 37°C for 45 minutes. The images were obtained by a confocal microscope (Olympus Corporation, FV1000, Tokyo, Japan). The mitochondria structures were observed.

Transmission Electron Microscopy for observing mitophagy

Observation of mitophagy in H9c2 cells under transmission electron microscopy. After centrifuged (5,000 ×g, 5 min, 4 °C), cells were harvested and resuspended in 2.5% glutaraldehyde, then were fixed overnight at 4 °C. After washed with buffer, the pellets were fixed in 100 mM cacodylate for 2.5 h. After washing with distilled water, the pellets were dehydrated with ethanol. It was embedded in resin at 65℃ for 48 hours. Then, the samples were cut into ultrathin sections and l loaded onto 300-mesh copper grids (Plano GmbH, Marburg, Germany), then stained with uranyl acetate and lead citrate. The grids were viewed under transmission electron microscopy (Hitachi, HT7800, Tokyo, Japan). (50)

Detection of reactive oxygen species (ROS) content and mitochondrial membrane potential (MMP) by laser confocal microscopy

Tetramethylrhodamine Ethyl Ester (TMRE) fluorescent dye (BestBio, BB-41053, Shanghai, China) was used to detect MMP in miR-130a OE and KO cells. 5×10⁵ cells were harvested and resuspended with 500 μl TMRE staining solution/mL. Then the cells were incubated for 15 minutes at 37 ° C. Nuclei were stained with DAPI (Abcam, ab104139, Cambridge, England). The cells were observed by laser confocal microscopy (Leica, STELLARIS 8, Weizler, Germany). Fluorescence intensity was measured by ImageJ software.

JC-1 dye (Thermo Fisher Scientific Inc, T3168, Waltham, MA) was used to detect MMP in GJA1 OE cells. JC-1 is a cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~525 nm) to red (~590 nm). Cells were dyed with JC-1 dye (2 μM in final concentration) and incubated at 37 °C, 5% CO₂ for 30 min. Nuclei were stained with DAPI (Abcam, ab104139, Cambridge, England). The cells were observed by laser confocal microscopy (Leica, STELLARIS 8, Weizler, Germany). Fluorescence intensity was measured by ImageJ software. Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio (ΔΨm).

ROS content was detected by Fluorometric Intracellular ROS Kit (Sigma-Aldrich Corporation, MAK145, St. Louis, MO). Cells were harvested and stained for 20 min at 37 ℃ according to the instructions of the Kit. Nuclei were stained with DAPI (Abcam, ab104139, Cambridge, England). The cells were observed by laser confocal microscopy (Leica, STELLARIS 8, Weizler, Germany). Fluorescence intensity was measured by ImageJ software.

Determination of mitophagy activity by co-localization experiment

Cells were incubated with mito-tracker green (Beyotime Biotechnology, C1048, Shanghai, China) and lyso-tracker red (Beyotime Biotechnology, C1046, Shanghai, China) at 37°C for 45 minutes, respectively. The images were obtained by a confocal microscope (Olympus Corporation, FV1000, Tokyo, Japan). The colocalization of mitochondria and lysosome structures was observed, and the Pearson’s correlation coefficient was used for quantitative analysis of the co-localization of lysosomal and mitochondrial. ImageJ software calculated the Pearson’s correlation coefficient.

Statistical analysis

Statistical analysis was conducted using GraphPad Prism 7 (Graphpad Software Inc., San Diego, CA). Data were expressed as mean ± standard deviation (SD), each of which represents at least three independent experiments. All data were first tested for normality via the Kolmogorov-Smirnov test and for equal variances via Levene’s test. Comparing continuous data variables among multi-group, we carried out an ordinary one-way ANOVA test and the two-group were analyzed by Student's t-test. The 95% confidence intervals (CI) for the parameters in our analysis were estimated and a two-sided P < 0.05 (the threshold) was defined as being statistically significant.

Conflict of Interest

The authors have no relevant financial or non-financial interests to disclose.

The authors have no competing interests to declare that are relevant to the content of this article.

All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.

The authors have no financial or proprietary interests in any material discussed in this article.

Availability of Data and Materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Author contributions

Xiao-jing Zhao and Yong-ming Yao contributed to the conception and design of the study. Yan Yan and Liu-yang Tian performed the experiments and acquired the data. Yang Han and Hui-ning Chen performed the statistical analysis. Yan Yan wrote the first draft of the manuscript. Sai-jia Cui wrote sections of the manuscript. Qian Jia provided helpful discussion and reviewed the manuscript. All authors contributed to the manuscript revision, read, and approved the submitted version.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (82001994, 82130062).

Ethics statement

Studies involving human participants adhered to the Declaration of Helsinki. Informed consent for participation in the study has been obtained, and this consent was in writing. The inclusion of human participants and the use of human data and human blood in this study were approved by the Ethical Committee for Medical Research of Chinese PLA General Hospital (approval number: S.2017-035-01). All animal experimental procedures followed the ARRIVE guidelines and were approved by the Animal Ethics Committee of the Chinese PLA General Hospital (approval number: 2017-X13-05).

1. Redd MA, Scheuer SE, Saez NJ, Yoshikawa Y, Chiu HS, Gao L, et al. Therapeutic inhibition of acid-sensing ion channel 1a recovers heart function after ischemia-reperfusion injury. Circulation 144, 947-60 (2021).

2. Mao XL, Cai Y, Chen YH, Wang Y, Jiang XX, Ye LP, et al. Novel targets and therapeutic strategies to protect against hepatic ischemia-reperfusion injury. Frontiers In Medicine 8, 757336 (2021).

3. Zhang GQ, Wang SQ, Chen Y, Fu LY, Xu YN, Li L, et al. MicroRNAs regulating mitochondrial function in cardiac diseases. Frontiers in Pharmacology 12, 663322 (2021).

4. Li G, Wang S, Fan Z. Oxidative stress in intestinal ischemia-reperfusion. Frontiers In Medicine 8, 750731 (2021).

5. Tang J, Zhuang S. Histone acetylation and DNA methylation in ischemia/reperfusion injury. Clinical Science 133, 597-609 (2019).

6. Song R, Hu XQ, Zhang L. Mitochondrial miRNA in cardiovascular function and disease. Cells 8, 1475 (2019).

7. Sygitowicz G, Sitkiewicz D. Mitochondrial quality control: The role in cardiac injury. Frontiers in Bioscience-Landmark 27, 96 (2022).

8. Gaal Z. Implication of microRNAs in carcinogenesis with emphasis on hematological malignancies and clinical translation. International Journal Of Molecular Sciences 23, 5838 (2022).

9. Xie Y, Hu J, Zhang X, Li C, Zuo Y, Xie S, et al. Neuropeptide Y induces cardiomyocyte hypertrophy via attenuating miR-29a-3p in neonatal rat cardiomyocytes. Protein and Peptide Letters 27, 878-887 (2020).

10. Sun T, Li MY, Li PF, Cao JM. MicroRNAs in cardiac autophagy: Small molecules and big role. Cells 7, 104 (2018).

11. Moghiman T, Barghchi B, Esmaeili SA, Shabestari MM, Tabaee SS, Momtazi-Borojeni AA. Therapeutic angiogenesis with exosomal microRNAs: An effectual approach for the treatment of myocardial ischemia. Heart Failure Reviews 26, 205-213 (2021).

12. Pakravan G, Foroughmand AM, Peymani M, Ghaedi K, Hashemi MS, Hajjari M, et al. Downregulation of miR-130a, antagonized doxorubicin-induced cardiotoxicity via increasing the PPARgamma expression in mESCs-derived cardiac cells. Cell Death & Disease 9, 758 (2018).

13. Wang H, Xu J, Wu X, Zheng S, Han Y, Ding G. Longitudinal change in microRNA-130a expression and its correlation with the risk of developing major adverse cardiovascular and cerebral events in patients undergoing continuous ambulatory peritoneal dialysis. Journal of Clinical Laboratory Analysis (2021). https://doi.org/10.1002/jcla.24039

14. Zhou T, Qin G, Yang L, Xiang D, Li S. LncRNA XIST regulates myocardial infarction by targeting miR-130a-3p. Journal of Cellular Physiology 234, 8659-8667 (2019).

15. Zheng T, Shi Y, Zhang J, Peng J, Zhang X, Chen K, et al. MiR-130a exerts neuroprotective effects against ischemic stroke through PTEN/PI3K/AKT pathway. Biomedicine & Pharmacotherapy 117, 109117 (2019).

16. Kuznetsov AV, Javadov S, Margreiter R, Grimm M, Hagenbuchner J, Ausserlechner MJ. The role of mitochondria in the mechanisms of cardiac ischemia-reperfusion injury. Antioxidants 8, 454 (2019).

17. Huang J, Wu R, Chen L, Yang Z, Yan D, Li M. Understanding anthracycline cardiotoxicity from mitochondrial aspect. Frontiers in Pharmacology 13, 811406 (2022).

18. Gu C, Yan J, Zhao L, Wu G, Wang YL. Regulation of mitochondrial dynamics by aerobic exercise in cardiovascular diseases. Frontiers in Cardiovascular Medicine 8, 788505 (2021).

19. Mangukia N, Rao P, Patel K, Pandya H, Rawal RM. Unveiling the nature's fruit basket to computationally identify Citrus sinensis csi-mir169-3p as a probable plant miRNA against Reference and Omicron SARS-CoV-2 genome. Computers in Biology and Medicine 146, 105502 (2022).

20. Shen Z, Chen Q, Jin T, Wang M, Ying H, Lu J, et al. Theaflavin 3,3'-digallate reverses the downregulation of connexin 43 and autophagy induced by high glucose via AMPK activation in cardiomyocytes. Journal Of Cellular Physiology 234, 17999-18016 (2019).

21. Wang X, Feng L, Xin M, Hao Y, Wang X, Shang P, et al. Mechanisms underlying astrocytic connexin-43 autophagy degradation during cerebral ischemia injury and the effect on neuroinflammation and cell apoptosis. Biomedicine & Pharmacotherapy 127, 110125 (2020).

22. Boengler K, Schulz R. Connexin 43 and mitochondria in cardiovascular health and disease. Advances in Experimental Medicine and Biology 982, 227-246 (2017).

23. Bell CL, Shakespeare TI, Smith AR, Murray SA. Visualization of annular gap junction vesicle processing: The interplay between annular gap junctions and mitochondria. International Journal of Molecular Sciences 20, 44 (2018).

24. Wang L, Li Q, Diao J, Lin L, Wei J. MiR-23a is involved in myocardial ischemia/reperfusion injury by directly targeting CX43 and regulating mitophagy. Inflammation 44, 1581-1591 (2021).

25. Xue J, Yan X, Yang Y, Chen M, Wu L, Gou Z, et al. Connexin 43 dephosphorylation contributes to arrhythmias and cardiomyocyte apoptosis in ischemia/reperfusion hearts. Basic Research in Cardiology 114, 40 (2019).

26. Martins-Marques T, Catarino S, Zuzarte M, Marques C, Matafome P, Pereira P, et al. Ischaemia-induced autophagy leads to degradation of gap junction protein connexin43 in cardiomyocytes. Biochemical Journal 467, 231-245 (2015).

27. Gustafsson AB, Gottlieb RA. Heart mitochondria: Gates of life and death. Cardiovascular Research 77, 334-343 (2008).

28. Pawlak A, Gewartowska M, Przybylski M, Kuffner M, Wiligorska D, Gil R, et al. Ultrastructural changes in mitochondria in patients with dilated cardiomyopathy and parvovirus B19 detected in heart tissue without myocarditis. Journal of Personalized Medicine 12, 177 (2022).

29. Diao RY, Gustafsson AB. Mitochondrial quality surveillance: Mitophagy in cardiovascular health and disease. American Journal of Physiology-Cell Physiology 322, C218-C230 (2022).

30. Salazar-Gonzalez H, Gutierrez-Mercado YK, Munguia-Galaviz FJ, Echavarria R. Signaling pathways involved in myocardial ischemia-reperfusion injury and cardioprotection: A systematic review of transcriptomic studies in Sus scrofa. Journal of Cardiovascular Development and Disease 9, 132 (2022).

31. Yau TH, Chong MH, Brigden ZM, Ngemoh D, Harky A, Bin Saeid J. The timing of surgical revascularization in acute myocardial infarction: When should we intervene? Journal of Cardiovascular Surgery 63, 179-186 (2022).

32. Zheng L, Wang Z, Li Z, Wang M, Wang W, Chang G. MicroRNA-130a inhibits proliferation of vascular smooth muscle cells by suppressing autophagy via ATG2B. Journal of Cellular and Molecular Medicine 25, 3829-3839 (2021).

33. Feng Y, Bao Y, Ding J, Li H, Liu W, Wang X, et al. MicroRNA-130a attenuates cardiac fibrosis after myocardial infarction through TGF-beta/Smad signaling by directly targeting TGF-beta receptor 1. Bioengineered 13, 5779-5791 (2022).

34. Chu X, Wang Y, Pang L, Huang J, Sun X, Chen X. miR-130 aggravates acute myocardial infarction-induced myocardial injury by targeting PPAR-gamma. Journal of Cellular Biochemistry 119, 7235-7244 (2018).

35. Colpaert RMW, Calore M. MicroRNAs in cardiac diseases. Cells 8, 737 (2019).

36. Dorn GW, 2nd. Evolving concepts of mitochondrial dynamics. Annual Review of Physiology 81, 1-17 (2019).

37. Tahrir FG, Langford D, Amini S, Mohseni Ahooyi T, Khalili K. Mitochondrial quality control in cardiac cells: Mechanisms and role in cardiac cell injury and disease. Journal Of Cellular Physiology 234, 8122-8133 (2019).

38. Vela D. Keeping heart homeostasis in check through the balance of iron metabolism. Acta Physiologica (2020). https://doi.org/10.1111/apha.13324

39. Yang M, He Y, Deng S, Xiao L, Tian M, Xin Y, et al. Mitochondrial quality control: A pathophysiological mechanism and therapeutic target for stroke. Frontiers in Molecular Neuroscience 14, 786099 (2021).

40. Pietzsch S, Ricke-Hoch M, Stapel B, Hilfiker-Kleiner D. Modulation of cardiac AKT and STAT3 signalling in preclinical cancer models and their impact on the heart. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1867, 118519 (2020).

41. Liu M, Wu Y. Role of mitophagy in coronary heart disease: Targeting the mitochondrial dysfunction and inflammatory regulation. Frontiers in Cardiovascular Medicine 9, 819454 (2022).

42. Denisenko TV, Gogvadze V, Zhivotovsky B. Mitophagy in carcinogenesis and cancer treatment. Discover Oncology 12, 58 (2021).

43. Basheer WA, Fu Y, Shimura D, Xiao S, Agvanian S, Hernandez DM, et al. Stress response protein GJA1-20k promotes mitochondrial biogenesis, metabolic quiescence, and cardioprotection against ischemia/reperfusion injury. JCI Insight (2018). https://doi.org/10.1172/jci.insight.121900

44. Li Y, Ge J, Yin Y, Yang R, Kong J, Gu J. Upregulated miR-206 aggravates deep vein thrombosis by regulating GJA1-mediated autophagy of endothelial progenitor cells. Cardiovascular Therapeutics 2022, 9966306 (2022).

45. Turkieh A, El Masri Y, Pinet F, Dubois-Deruy E. Mitophagy regulation following myocardial infarction. Cells 11, 199 (2022).

46. Li Q, Liu Y, Huang Q, Yi X, Qin F, Zhong Z, et al. Hypoxia acclimation protects against heart failure postacute myocardial infarction via Fundc1-mediated mitophagy. Oxidative Medicine and Cellular Longevity 2022, 8192552 (2022).

47. Zhang W. The mitophagy receptor FUN14 domain-containing 1 (FUNDC1): A promising biomarker and potential therapeutic target of human diseases. Genes & Diseases 8, 640-654 (2021).

48. Mendis S, Thygesen K, Kuulasmaa K, Giampaoli S, Mahonen M, Ngu Blackett K, et al. World health organization definition of myocardial infarction: 2008-09 revision. International journal of epidemiology 40, 139-146 (2011).

49. Lewis NC, Bailey DM, Dumanoir GR, Messinger L, Lucas SJ, Cotter JD, et al. Conduit artery structure and function in lowlanders and native highlanders: Relationships with oxidative stress and role of sympathoexcitation. Journal of Physiology 592, 1009-1024 (2014).

50. Liu B, Yan Y, Wang X, Chen N, Wu J. Locally generated C3 regulates the clearance of Toxoplasma gondii by IFN-gamma-primed macrophage through regulation of xenophagy. Frontiers in Microbiology 13, 944006 (2022).

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MiR-130a-3p Regulates FUNDC1-Mediated Mitophagy by Targeting GJA1 in Myocardial Ischemia/Reperfusion Injury

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