Metformin confers longitudinal cardiac protection by preserving mitochondrial homeostasis following myocardial ischemia/reperfusion injury

Myocardial ischemia–reperfusion (I/R) injury is associated with systemic oxidative stress, cardiac mitochondrial homeostasis, and cardiomyocyte apoptosis. Metformin has been recognized to attenuate cardiomyocyte apoptosis. However, the longitudinal effects and pathomechanism of metformin on the regulation of myocardial mitohormesis following I/R treatment remain unclear. This study aimed to investigate the longitudinal effects and mechanism of metformin in regulating cardiac mitochondrial homeostasis by serial imaging with the 18-kDa translocator protein (TSPO)–targeted positron emission tomography (PET) tracer 18F-FDPA. Myocardial I/R injury was established in Sprague–Dawley rats, which were treated with or without metformin (150 mg/kg per day). Serial gated 18F-FDG and 18F-FDPA PET imaging were performed at 1, 4, and 8 weeks after surgery, followed by analysis of ventricular remodelling and cardiac mitochondrial homeostasis. The correlation between Hsp60 and 18F-FDPA uptake was analyzed. After PET imaging, the activity of antioxidant enzymes, immunostaining, and western blot analysis were performed to analyze the spatio-temporal effects and pathomechanism of metformin for cardiac protection after myocardial I/R injury. Oxidative stress and apoptosis increased 1 week after myocardial I/R injury (before significant progression of ventricular remodelling). TSPO expression was correlated with Hsp60 expression and was co-localized with inflammatory CD68+ macrophages in the infarct area, and TSPO uptake was associated with an upregulation of AMPK-p/AMPK and a downregulation of Bcl-2/Bax. However, these effects were reversed with metformin treatment. Eight weeks after myocardial I/R injury (representing the advanced stage of heart failure), 18F-FDPA uptake in myocardial cells in the distal non-infarct area increased without CD68+ expression, whereas the activity decreased with metformin treatment. Taken together, these results show that a prolonged metformin treatment has pleiotropic protective effects against myocardial I/R injury associated with a regional and temporal dynamic balance between mitochondrial homeostasis and cardiac outcome, which were assessed by TSPO-targeted imaging during cardiac remodelling.


Introduction
Myocardial ischemia-reperfusion (I/R) injury causes severe manifestations, including left ventricular remodelling and cardiac dysfunction, which ultimately exacerbate, leading to heart failure [1,2]. Oxidative metabolism in the mitochondria is the main source of cardiac energy consumption, and mitochondrial dysfunction is considered the primary mechanism linking cardiac contractile failure after myocardial I/R injury [3]. The mammalian mitochondrial permeability transition pore (mPTP) is essential for maintaining This article is part of the Topical Collection on Cardiology.
Jing Tian and Yaqi Zheng have contributed equally to the manuscript. mitochondrial homeostasis and membrane potential [4]. The irreversible myocardial damage following myocardial I/R injury has been proposed to involve mPTP opening. Thus, myocardial I/R injury appears to be closely linked to mitochondrial impairment. Moreover, excessive production of reactive oxygen species (ROS) is involved in several detrimental processes, including cell apoptosis triggered by the opening of the mPTP [5]. Indeed, enhanced ROS production is related to microvascular pathologies under myocardial ischemia conditions. Excessive ROS generally causes an inflammatory response by disrupting mitochondrial membrane homeostasis [6]. Therefore, therapeutic strategies for I/R injury have been developed targeting mPTP inhibition to modulate energy homeostasis, mitochondrial function, and ROS production in cardiomyocytes.
One such strategy may involve the repurposing of metformin, which is widely used to treat type 2 diabetes and also has a cardiac-protective mechanism that is proposed to be related to the mitochondria-mediated modulation of energy homeostasis. Metformin has been reported to activate AMP-activated protein kinase (AMPK), an intracellular energy sensor in intact cardiomyocytes [7,8]. Several studies corroborated that metformin may regulate mitochondrial homeostasis by restoring myocardial AMPK and reducing oxidative stress [9,10]. However, the underlying mechanism and longitudinal cardio-protection effects of metformin in association with mitohormesis have not been elucidated.
There is increasing recognition of the need for specific diagnostic biomarkers of myocardial I/R injury that can accurately assess myocardial death progression from a stress injury. Mitochondria impairment becomes a major biomarker for ischemic cardiac injury [11]. Hsp60 is a predominantly mitochondrial matrix protein, which is induced by mitochondrial stress/impairment, and it plays a critical role in the regulation of mitochondrial function [12].
Moreover, translocator protein (TSPO) is an 18-kDa protein, as a key component of the mPTP, which plays an essential role in regulating innate immunity [13]. TSPO is mainly expressed on the mitochondrial membrane [14], which is involved in the pathophysiological processes of mitochondrial homeostasis, apoptosis, and oxidative stress. In addition, upregulation of TSPO expression was found to be associated with the accumulation of ROS and the breakdown of mitochondrial homeostasis. Combined with such markers, advances in molecular imaging are expected to help identify myocardial I/R injury and facilitate timely intervention with effective therapies to prevent cardiac deterioration. Fluorine-18-labelled N,N-diethyl-2-(2-(4-(2-fluoroethoxy) phenyl)-5,7-dimethylpyrazolo [1,5-a]pyrimidin-3-yl)acetamide ( 18 F-FDPA) was developed as a novel TSPO-specific radioligand, which can be used to detect activated microglia in neurodegenerative disorders [15,16]. Our previous study [17] indicated that the increased cardiac uptake of 18 F-FDPA in rats with acute myocardial infarction at 1 week after surgery was related to the infiltration of inflammatory cells. Nevertheless, the utility of 18 F-FDPA in TSPO imaging for quantification of the degree of cardiac mitochondrial homeostasis after myocardial I/R injury has not been assessed.
Accordingly, the primary aim of the present study was to investigate the spatio-temporal effects and pathomechanism of metformin for cardiac protection after myocardial I/R injury. Specifically, we serially evaluated in vivo myocardial TSPO expression in correlation with the extent of mitochondrial homeostasis and cardiac function in a rat model of myocardial I/R injury using 18 F-FDPA positron emission tomography (PET) imaging.

Animal model
Thirty-six male Sprague-Dawley rats (280-300 g) were purchased from SPF Biotechnology Co., Ltd. (Beijing, China) and underwent transient coronary artery ligation to induce myocardial I/R or sham surgery as previously described [18,19]. In brief, the rats were anaesthetized with isoflurane (4.0% induction, 2.0% maintenance) and mechanically ventilated with a tidal volume of 8.0-13.0 ml/kg, a respiratory rate of 80 breaths per minute, and an inspiratory-toexpiratory ratio of 1:2. A 7-0 polypropylene suture with a curved needle was placed under the origin of the left anterior descending (LAD) artery and a medical latex tube was placed over the artery. Myocardial ischemia was induced by tightening the suture for 30 min. Successful induction of myocardial ischemia was verified by the presence of STsegment elevation on electrocardiography. The suture was then loosened to restore coronary circulation, ensuring that the myocardium turned grey after LAD ligation. Twentyfour rats with I/R were randomly divided into two groups: the vehicle (0.9% saline) group and metformin group. The rats in the metformin group received metformin (150 mg/ kg per day) every day for 8 weeks based on previous experiments [20,21]. The other 12 rats were selected as the sham group. Sham-operated animals underwent the same surgical procedures but were not subjected to LAD coronary artery ligation. Body weight and blood glucose levels were measured at designated times. The experimental protocols are schematically displayed in Fig. 1.
According to the scheduled time point, animals were euthanized with an overdose of isoflurane (5.0% isoflurane with 2.0 L/min oxygen) followed by cervical dislocation. All animal experiments were performed according to Beijing's laboratory animal management regulations and were approved by the Animal Care Committee of Capital Medical University (Ethical approval number: AEEI-2019-167). 18 F-FDPA was automatedly synthesized by the CFN-MPS200 module (Sumitomo Heavy Industries, Ltd., Tokyo, Japan) as described previously [17]. The radiochemical purity was > 99%. The molar activity was 273-532 GBq/μmol.

Whole-body small-animal PET/computed tomography (CT)
PET/CT imaging was performed with a dedicated micro-PET/CT scanner (Inveon PET/CT; Siemens, Germany). Each rat was individually placed into a chamber connected to an isoflurane anaesthesia unit, and anaesthesia was induced using an airflow rate of 2.0 L/min containing 4.0% isoflurane. The animal was then immediately placed in a prone position on the scanning bed, and the airflow rate was reduced to 0.8-1.5 L/min with 2-2.5% isoflurane. Approximately 74 MBq 13 N-NH 3 was injected via the tail vein, and a dynamic 15-min (2 frames: 1 × 300 s, 1 × 600 s) image was acquired in list mode, followed by a 2-beds CT scan using the "magnification low" acquisition setting (projection: 180; binning: 4 × 4; transaxial field of view: 53.9 mm; axial scanning length: 134 mm; voltage: 80 kV; current: 500 μA). Four hours after 13 N-NH 3 image acquirement, 29.6-33.3 MBq 18 F-FDG was injected via the tail vein. Forty minutes later, a 30-min gated PET scan was performed, followed by a CT scan following the same method described above. The next day, 18 F-FDPA (30.0-35.0 MBq) was injected into the same rat, and a static 15-min PET image was acquired at 40 min post injection, followed by a CT scan according to the same method described above.

Image analysis
Images were analyzed by two experienced nuclear cardiologists independently using Inveon Research Workplace software (Siemens) and the cardiac PMOD software 4.2 package (PMOD Technologies Ltd., Zurich, Switzerland). Heart analysis was performed as described previously [15,[22][23][24]. 13 N-NH 3 perfusion imaging was assessed using an AHA 17-segment model according to the American Society of Nuclear Cardiology guidelines [25]. Each segment accounts for 6% of the left ventricle (LV). The total perfusion defect (TPD; percentage of the LV myocardium) was defined using a threshold (< 60%) of the normalized maximum signal of 13 N-NH 3 uptakes [25,26].
The regional changes in 18 F-FDPA uptake were automatically determined using the PMOD software in each vessel (LAD, LCx, and RCA), respectively. For quantification of 18 F-FDPA uptake in the whole myocardium, myocardium in the infarct territory, and remote non-infarct territory of the inferior wall, regions of interest (ROIs) in the 13 N-NH 3 polar maps were delineated and copied to the 18 F-FDPA polar maps to allow for precise, spatially matched quantification of 18 F-FDPA activity. To account for differences in tracer delivery, the regional or global mean standardized uptake value (SUVmean) of 18 F-FDPA in the LV and that in the left atrium (LA) were analyzed. The global targetto-background ratio (TBR) in the whole heart was calculated as the global SUVmean/LA SUVmean, whereas the regional TBR was calculated as the regional SUVmean/ LA SUVmean.
To assess the myocardial systolic and diastolic function, electrocardiogram-gated 18 F-FDG was conducted serially at 1 week, 4 weeks, and 8 weeks post-I/R injury. The cardiac PMOD software was used to quantitatively measure the LV end-diastolic volume (LVEDV), end-systolic volume (LVESV), and LV ejection fraction (LVEF).

Oxidative stress measurement
The concentrations of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) in the rat serum were measured using corresponding enzymelinked immunosorbent assay kits from the Beijing Sinouk Institute of Biological Technology (Beijing, China) according to the manufacturer's protocols.

Western blot analysis
Western blot analysis was performed as described previously [28]. In brief, the protein concentration was measured using a bicinchoninic acid protein assay kit (YTHXBio, China).

Statistical analysis
Data with a normal distribution are expressed as the mean ± standard deviation, whereas non-normally distributed data are expressed as the median and interquartile range. The mean values of continuous variables were compared between two groups using Student's t-test with normality or the Mann-Whitney U test without normality. Multiple groups were compared by analysis of variance followed by post hoc testing with normality (Tukey's post hoc tests with equal variance assumed or Dunnett's T3 test without equal variance assumed) or the Kruskal-Wallis H test without normality. The Pearson correlation was used to analyze the relationship between Hsp60 and 18 F-FDPA uptake. P < 0.05 was defined as statistically significant. All statistical analyses were performed using SPSS software, version 26.0 (SPSS Inc., Chicago, IL, USA) and Prism 7 software (GraphPad).

Animal body weight and blood glucose levels
Body weight and fasting blood glucose in the metformin group did not differ from the vehicle group at 8 weeks (all P > 0.05) (Supplementary Table 1).

Metformin improved LV structure and function after myocardial I/R injury
Serial 13 N-NH 3 perfusions showed that the extent of TPD in the vehicle group exhibited a 10% decrease in the LV at 1 week after I/R injury ( Fig. 2A). There was no significant difference in the TPD between the vehicle group and metformin group over 8 weeks ( Fig. 2A).
Functional analysis by serial gated 18 F-FDG PET/CT revealed no significant changes in the LVEF in the sham group over 8 weeks (Fig. 2B). After I/R injury, the rats in both the vehicle and metformin groups exhibited marked LV remodelling during follow-up, as attested by significant increases in the LVESV (Fig. 2D) and significant decreases in the LVEF (Fig. 2B) compared with those of the sham group. Notably, the LVEF in the metformin group was higher than that in the vehicle group and approached the normal level at 8 weeks, although the difference was not statistically significant, suggesting that the mechanisms underlying the response to metformin therapy are largely related to LV systolic function.

Metformin prevented oxidative stress in rats after I/R injury during heart failure progression
After I/R injury, the metformin group exhibited an anti-oxidative stress response during follow-up, as attested by significant increases in the SOD and GSH-Px levels (all P < 0.01) (Fig. 3A, C), and decreases in the MDA level (all P < 0.05) (Fig. 3B) compared with those of the vehicle group.

Regional mitochondrial homeostasis in rats after I/R injury during heart failure progression
The Hsp60 in the remote territory was higher than that in the infarct territory 1 week after I/R injury (P < 0.01) ( Fig. 4A-C). Overall, mitochondrial Hsp60 was strongly associated with TSPO expression ( 18 F-FDPA uptake) in the remote territory (r = 0.797, P < 0.001) and was weakly associated in the infarct territory (r = 0.439, P = 0.032) at 1 week after I/R injury (Fig. 4D, E).

Metformin maintained mitochondrial homeostasis in rats after I/R injury in a time-dependent manner
The normalized SUV ratios of global cardiac 18 F-FDPA uptake to the LA (global TBR) and the remote territory TBR in the vehicle group were markedly elevated at 4 and 8 weeks compared with those of the sham group (all P < 0.01) (Fig. 5A, B), respectively. The TBR in the infarct territory was significantly lower than that in the remote territory at 1, 4, and 8 weeks after I/R injury (all P < 0.01) (Fig. 5C). Thus, this semi-quantitative analysis demonstrated that TSPO was overexpressed in the remote non-infarct  . The correlations between Hsp60 and TSPO signal in the infarct territory (r = 0.439, P = 0.032) (D) and in the remote territory (r = 0.797, P < 0.001) (e) at the acute phase after I/R injury. P values are based on the Student's t-test, ** P < 0.01 between the groups. Pearson correlation was used for statistical analysis territory of the myocardium rather than in the infarct territory of the myocardium in a time-dependent manner. After I/R injury, the metformin group showed a significant decline in the TBRs of the global, infarct, and remote territory portions of the myocardium at 8 weeks compared with those of the vehicle group (Fig. 6A-D). 18 F-FDPA segmental distribution results showed that the TBR of the metformin group in the vascular territory LAD was significantly lower than that of the sham group at 1 week and 8 weeks after I/R injury (P < 0.01) (Supplementary Table 2).

Effects of metformin on the infarct territory at 1 week after I/R injury
Haematoxylin and eosin staining showed a massive accumulation of inflammatory cells in the infarct territory in the vehicle group at 1 week after I/R injury (Fig. 7A, i). Immunofluorescence confirmed that the elevated TSPO signal in the infarct territory co-localized with abundant CD68 + inflammatory cells in both the vehicle and metformin groups (Fig. 7B, iii). To assess the presence of regional cell survival and the AMPK-dependent regulatory pathway in mitochondrial homeostasis, we evaluated the relative protein expression levels of Bcl-2/Bax and AMPK-p/AMPK by western blot analysis. The levels of Bcl-2/Bax in the vehicle group exhibited an 89% (P = 0.001) decrease compared with those of the sham group in the infarct territory 1 week after I/R injury. However, this decrease in the levels of Bcl-2/Bax was prevented in the metformin group (Fig. 7C). Notably, the levels of AMPK-p/AMPK in the vehicle group showed a sixfold increase (P = 0.001) compared with those of the sham group. Nevertheless, this increase in the levels of AMPKp/AMPK was abrogated in the metformin group (Fig. 7E). These results suggest that metformin protects against I/Rinduced apoptosis by inhibiting endogenous AMPK in the infarct territory at the acute phase.

Effects of metformin on the remote non-infarct territory at 1 week after I/R injury
Without CD68 + co-localization, TSPO staining was consistent with the presence of non-inflammatory cells in to-background ratio (TBR) = the global SUVmean/LA SUVmean. Regional TBR = the regional SUVmean/LA SUVmean. N = 9 independent experiments. LA, left atrium; vehicle, saline. Data are presented as a box plot (box, 25-75% interquartile range; centre line, median) overlaid with a dot plot (individual data points). ** P < 0.01, *** P < 0.001 (Mann Whitney U test) the remote non-infarct territory at 1 week after I/R injury (Fig. 7A, B; ii). In parallel, the increase in the TSPO signal was reduced with metformin treatment (Fig. 7A, B; iv). Western blot analysis showed that the levels of Bcl-2/Bax in the vehicle group were 84% (P < 0.001) lower than those in the sham group in the remote non-infarct territory at 1 week after I/R injury, and this decrease was prevented in the metformin group (Fig. 7D). However, there was no significant difference in the levels of AMPK-p/AMPK between the vehicle and metformin groups (Fig. 7F).

Effects of metformin on the infarct territory at 8 weeks after I/R injury
Without CD68 + co-localization, TSPO staining was consistent with non-inflammatory cells in the infarct territory at 8 weeks Fig. 6 Metformin maintains mitochondrial homeostasis after myocardial ischemia-reperfusion (I/R) injury. Representative TSPO polar maps of the three groups over 8 weeks following I/R injury (A). The TSPO signal is shown for the sham group (top row), vehicle group (middle row), and metformin group (bottom row) at 1, 4, and 8 weeks after I/R injury. All serial images are scaled identically. Semi-quantitative analysis of the TSPO signal in the global (B), infarct territory (C), and remote non-infarct territory (D) of I/R in the three groups over 8 weeks following I/R injury (or sham surgery). Global target-tobackground ratio (TBR) = the global SUVmean/LA SUVmean. Regional TBR = the regional SUVmean/LA SUVmean. N = 9 independent experiments. LA, left atrium; Vehi, saline; Met, metformin. Data are presented as a box plot (box, 25-75% interquartile range; centre line, median). * P < 0.05, ** P < 0.01 vs. I/R 1wk (Mann Whitney U test and Kruskal-Wallis test with Dunn's post hoc test) Fig. 7 Metformin protects against ischemia-reperfusion (I/R)-induced mitochondrial dysfunction and apoptosis by inhibiting endogenous AMPK expression in the infarct territory at acute phase. (A) Haematoxylin and eosin (H&E) staining to define the general myocardial morphology and local position for the acute infarct territory (i, iii) and remote territory (ii, iv) in the vehicle and metformin groups at 1 week after I/R injury. (B) Gross histological sections stained with H&E indicate the position of inflammation under a light microscope (magnification 40 × , scale bar = 20 μm) (top row). Confocal fluorescence microscopy with dual fluorescent staining for CD68 (green, second row) and TSPO (red, third row). Blue fluorescence of diamidinephenylindole (DAPI) identifies the nuclei in the fused images (bottom row). Western blot analysis and quantification for apoptosis-related proteins Bcl-2/Bax in the infarct territory (C) and remote territory (D) (N = 3 per group). Western blot analysis and quantification of AMPK pathway-related proteins AMPK-p/AMPK in the infarct territory (E) and remote territory (F) (N = 3 per group). Vehi, saline; Met, metformin. P values are based on the Student t-test after I/R injury (Fig. 8A, B; i). In parallel, the increase in the TSPO signal was reduced with metformin treatment (Fig. 8A,  B; iii). Western blot analysis showed that the levels of Bcl-2/ Bax in the vehicle group were 73% (P < 0.001) lower than those in the sham group in the infarct territory at 8 weeks after I/R injury. However, this decrease in Bcl-2/Bax levels was prevented in the metformin group (Fig. 8C). There was no significant difference in the levels of AMPK-p/AMPK between the vehicle and metformin groups (Fig. 8E).

Effects of metformin on the remote non-infarct territory at 8 weeks after I/R injury
Without CD68 + co-localization, TSPO staining was consistent with the non-inflammatory cells in the remote non-infarct territory at 8 weeks after I/R injury (Fig. 8A, B; ii). In parallel, the increase in the TSPO signal was lower with metformin treatment (Fig. 8A, B; iv). Western blot analysis showed that the levels of Bcl-2/Bax in the vehicle group were 35% (P = 0.028) lower than those in the I/R + sham group in the remote noninfarct territory at 8 weeks after I/R injury. A significant increase in the Bcl-2/Bax ratio was observed in the metformin group as compared with that of the vehicle group (Fig. 8D). The AMPK-p/AMPK ratio in the vehicle group exhibited a 93% (P < 0.001) decrease compared with that in the sham group, consistent with the pattern for Bcl-2/Bax expression. However, this decrease in the AMPK-p/AMPK ratio was prevented in the metformin group (Fig. 8F). Thus, metformin protected against I/R-induced apoptosis by promoting exogenous AMPK in the remote territory at the chronic phase.

Discussion
Severe and extensive myocardial injury leads to cellular metabolic disorder, which disrupts ATP synthesis in the respiratory chain. Subsequently, ROS production is enhanced while the threshold for the opening of the mitochondrial permeability transition pore (mPTP) is reduced which induces an imbalance of myocardial mitochondrial homeostasis and eventually results in cell death [29]. Consistently, in this study, we found that with aggravation of the myocardial I/R-induced damage response, the levels of SOD and GSH-Px in the blood gradually decreased, the content of MDA gradually increased, and the in vivo TSPO expression in the whole heart gradually increased. The results suggest that the extent of the generation of ROS and mitochondrial homeostasis imbalance were time-dependent and related to the severity of myocardial I/R injury. Hsp60 has been known as a molecular chaperonin and assists in the folding of imported proteins to native conformation, which is a biomarker for mitochondrial homeostasis [30]. According to our previous study, 18 F-FDPA uptake was inhibited by PK11195, which demonstrated that 18 F-FDPA is a high-affinity ligand of TSPO [17]. Moreover, we showed a co-localization and correlation between Hsp60 expression and 18 F-FDPA activity in the infarcted territory after acute I/R injury, indicating a promising sensitivity and specificity of TSPO targeting to mitochondria density.
Metformin is a drug used for the clinical treatment of diabetes, which can activate AMPK by increasing the AMP/ATP ratio in cells and upregulate the antioxidant enzyme SOD to reduce myocardial I/R injury [31]. After intervention with metformin, with the prolongation of treatment time, the serum SOD and GSH-Px levels of the rats gradually increased, and the MDA content gradually decreased. The 18 F-FDPA uptake in the global heart in vivo PET imaging was reduced. Taken together, these results indicate that metformin could enhance the activity of antioxidant enzymes and maintain myocardial mitochondrial homeostasis after I/R injury.
Endogenous AMPK activation and mitochondrial homeostasis imbalance are important factors in promoting apoptosis in myocardial I/R injury [32,33]. Previous studies have demonstrated that under stress conditions, the macrophage migration inhibitory factor (MIF) upregulates and activates a variety of receptors on macrophages [34,35], and eventually causes myocardial cell apoptosis. In this study, at 1 week after myocardial I/R injury, AMPK-p/AMPK protein expression was upregulated, whereas Bcl-2/Bax protein expression was downregulated, and TSPO and CD68 + (a macrophage marker) co-localized in the infarct area with high expression. These results suggest that activation of endogenous AMPK and the imbalance of mitochondrial homeostasis in the infarct area under acute stress are related to the inflammatory response in this area. Further analyses showed that compared with the vehicle group, myocardial AMPK-p/AMPK protein expression in the infarct area of the metformin group was downregulated, the myocardial TSPO in vivo expression level decreased, and Bcl-2/Bax protein expression was upregulated. These results suggest that metformin could maintain mitochondrial homeostasis and attenuate cardiomyocyte apoptosis by inhibiting endogenous myocardial AMPK activation in the infarct area before significant progression of LV remodelling.
At 8 weeks after myocardial I/R injury, the in vivo TSPO expression level in cardiomyocytes in the distal non-infarcted area increased, no CD68 + inflammatory cells were detected, and the expression levels of AMPKp/AMPK and Bcl-2/Bax proteins decreased. These results suggest that mitochondrial homeostasis imbalance and cardiomyocyte apoptosis are present in the area distal to the infarct at the late stage of heart failure, despite the lack of endogenous AMPK activation or CD68 + cell accumulation in myocardial inflammatory cells in this area. Thackeray et al. [15] also reported no accumulation of CD68 + cells in the high-TSPO expression area distal to the infarct at the late stage of heart failure, which was speculated to be related to mitochondrial dysfunction. In Fig. 8 Metformin protects against ischemia-reperfusion (I/R)-induced mitochondrial dysfunction and apoptosis by promoting exogenous AMPK expression in the remote territory at the chronic phase after injury (8 weeks). (A) Haematoxylin and eosin (H&E) staining to define the general myocardial morphology and local position for the chronic infarct territory (i, iii) and remote territory (ii, iv) in the vehicle and metformin groups at 8 weeks after I/R injury. (B) Gross histological sections stained with H&E indicate the positioning of inflammation under a light microscope (magnification 40 × , scale bar = 20 μm) (top row). Confocal fluorescence microscopy with dual fluorescent staining for the monocyte marker CD68 (green, second row) and the imaging target TSPO (red, third row). Blue fluorescence of diamidine-phenylindole (DAPI) identifies the nuclei in the fused images (bottom row). Western blot analysis and quantification for apoptosis-related proteins Bcl-2/Bax in the infarct territory (C) and remote territory (D) (N = 3 per group). Western blot analysis and quantification of AMPK pathway-related proteins AMPK-p/AMPK in the infarct territory (E) and remote territory (F) (N = 3 per group). Vehi, saline; Met, metformin. P values are based on the Student t-test the present study, metformin was used for intervention in myocardial I/R injury, which further decreased the activity of 18 F-FDPA in the distal non-infarct area, the expression of AMPK-p/AMPK and Bcl-2/Bax proteins was increased in parallel. These results further confirm the protective effects of metformin on mitochondrial function in the distal non-infarct area.
TSPO is involved in modulating mPTP opening/closure, leading to either apoptotic cell death via mPTP opening or cell protection mediated by mPTP blocking, and hence regulating mPTP induced apoptosis [36]. Notably, our results show that metformin treatment retards myocyte apoptosis by the determination of Bcl-2 expression, which is a biomarker for cellular apoptosis. Moreover, TSPO showed constant regulation, which might indicate the underlying physiological mechanism between TSPO expression and the apoptosis process. Downregulation of TSPO prevents mPTP opening and protects against the inflammation-related cell death in the infarct territory at the acute phase, which has been related to the transient inhibition of mitochondrial permeability transition-mediated necrosis. However, we speculated that even without inflammatory cell infiltration, persistent mitochondrial outer membrane permeabilization exacerbates I/R-induced apoptosis by releasing free oxygen radicals in the chronic phase.
In addition, it is also known that systemic oxidative stress has been shown to affect the degree of repair of the damaged myocardium via paracrine action. Following myocardial infarction, the myocardium in the distal non-infarct area repairs the myocardial structure via the paracrine pathway, limiting LV expansion and protecting LV function [37]. Consistently, we found that compared with the vehicle group, the LVESV decreased and the LVEF increased in the metformin group, though there was no significant difference between the two groups. This finding suggests that the protective effects of metformin against myocardial injury at the late stage of heart failure might be related to changes in the microenvironment in the distal non-infarct area, which then activate AMPK expression through the paracrine pathway, improve myocardial cell viability in this area and limit LV dilatation, and ultimately protect LV function. In the present study, we utilized gated PET/CT imaging to serially evaluate the changes in LV remodelling and the LVEF. Using this modality, we demonstrated that metformin therapy significantly improved LV function and inhibited LV remodelling.
Some limitations of the present study should be considered. First, based on our preliminary experiment and a previous study, a single metformin dose was selected for the present study. Second, the efficacies of metformin need to be furtherly validated in diabetic rats. Therefore, further validation on diabetic animals is warranted. Third, in the present study, we performed static-alone acquisition of 18 F-FDPA after 40 min according to our previous experimental results, which demonstrated that 18 F-FDPA accumulation reaches an equilibrium after 30-min circulation time in the heart, lungs, spleen, kidneys, and intestine in normal rats. Dynamic modelling of 18 F-FDPA cardiac uptake is warranted in future studies. Finally, our results also suggest that 18 F-FDPA PET imaging in chronic heart failure can provide a surrogate measure of mitochondrial homeostasis, which may help predict LV remodelling and assess the response to heart failure therapies. However, TSPO tracer uptake by the mitochondria-rich myocardial tissue could impact the resolution of inflammation against non-inflammatory signals in the early stage of I/R injury. Therefore, additional studies are needed to investigate the clinical implications of our findings.

Conclusion
In summary, this study is the first to demonstrate that a prolonged administration of metformin exerted a pleiotropic protective effect against myocardial I/R injury, which was associated with a temporal and regional dynamic balance between mitochondrial homeostasis and cell death, as assessed by TSPO-targeted in vivo imaging during cardiac remodelling. Specifically, metformin attenuated the apoptosis of injured cardiomyocytes via an endogenous pathway at the early phase of I/R injury. Subsequently, prolonged treatment of metformin promoted mitochondrial homeostasis of remote cardiomyocytes at the chronic phase following I/R.