Inhibiting mTOR enhanced Cardiac STAT3 phosphorylation at site Ser 727 and attenuated Myocardial Ischemia Reperfusion Injury in diabetic rats

: Background : Reduced levels of myocardial STAT3 activity in diabetic hearts may contribute to the increased susceptibility to ischemia-reperfusion injury (I/RI). The protein mammalian target of rapamycin (mTOR) can regulate metabolism and cell processes and plays major roles in the dynamics of I/RI. However, the role of mTOR in the regulation of myocardial STAT3 and thereby its impact on myocardial I/RI in diabetes at relatively late stages of the disease is unknown. Methods: Diabetes was induced by Streptozotocin in Sprague-Dawley rats. Myocardial I/RI was achieved with coronary occlusion for 30 minutes and reperfusion for 2 hours in absence or presence of the mTOR inhibitor rapamycin. In vitro cardiomyocyte hypoxia/re-oxygenation (H/R) was established in H9C2 cells. Results : In diabetic rats, the levels of troponin-I (Tn-I), lipid peroxidation products 15-F2t-Isoprostane (15-F2t-Iso) and malondialdehyde (MDA), and the protein expression of mTOR were all significantly increased ， while SOD release, the level of phosphorylated STAT3(p-STAT3-Ser727) were both significantly decreased compared to non-diabetic rats. Myocardial I/RI significantly increased the infarct size (IS) and further increased the mTOR activation and decreased p-STAT3-Ser727 compared to diabetic rats. The selective mTOR inhibitor rapamycin reversed these changes and conferred cardioprotective effect. In H9C2 cells, high glucose (HG) significantly increased lactic dehydrogenase (LDH) release, apoptosis cells, ROS production and the activation of mTOR, but decreased p-STAT3-Ser727. H/R further increased cellular injury, mTOR gene knock-down significantly reduced H/R injury. Conclusion : Myocardial mTOR was enhanced in diabetes and contributed to I/RI. mTOR inhibition attenuated myocardial I/RI through increasing p-STAT3-Ser727. the inhibitor of STAT3 or cardiac specific STAT3 knock out to observe the role of STAT3 in diabetic myocardial I/RI. In our another study exploring the effe cts of ischemia preconditioning (RIPC), we demonstrated that STAT3 gene


Introduction:
Myocardial ischemia reperfusion injury (I/RI) is a leading cause of death especially for subjects with diabetes, but its underlying mechanisms are largely unknown. Myocardial infarction is irreversible and is the characteristic consequence of sustained myocardial ischemia with or without reperfusion. Increasing duration of ischemia causes progressive irreversible injury and thus, reducing infarct size is the first goal for I/RI protection. The pathophysiology of I/RI mainly includes autophagy, necrosis, apoptosis, necroptosis, pyroptosis and ferroptosis [1]. A great number of signaling molecules participated in I/RI process, for example, autacoids, such as adenosine and bradykinin; lipid molecules，and these have closed relationship with protein STAT3, a key protein in the cardiac pro-survival RISK and SAFE pathways.
These two signaling pathways correlates with each other and play central roles in I/RI and ischemia preconditioning [2]. The mammalian target of rapamycin (mTOR) protein is a 289 KD serine/threonine kinase, which can regulate cell growth and survival [3], mTOR also plays important roles and functions as nutrient energy sensor facilitating the regulation of autophagy [4]. With respect to the heart, mTOR is a necessary component for the physiological regulation of functions related to cardiac structure and cardiac metabolism [3]. mTOR also facilitates completion of maintenance of normal microvascular barrier functions and endothelial permeability.
Previous research has indicated that the PI3K/Akt/mTOR signaling pathway is mediated by insulin [4]. The pathway related to inhibition signaling for GSK-3 , the mTOR-dependent angiogenesis signaling pathway, and the mTOR activationsignaling pathway are major signaling pathways related to cardioprotection [5]. Das et al. reported that application of the mTOR inhibitor of rapamycin (0.25 mg/kg, i.p.) prior to ischemia consequently reduced I/RI in mice by inducing the activation of the janus kinase 2 (JAK2) Signal Transducer and Activator of Transcription signaling (STAT3). These findings demonstrated that mTOR was a possible regulator of STAT3. STAT3 has also been found to play central roles in maintaining cardiomyocyte function, modulating conditions in the cardiac microenvironment, and is known to communicate with cardiac fibroblasts [6,7]. STAT3 participated in myocardial preconditioning, and the phosphorylation of STAT3 at sites of tyrosine705 and serine727 have been shown to confer cardioprotection. In Heusch et al studies, they demonstrated the STAT3 phosphorylation at tyrosine705 was significantly greater at 120 minutes reperfusion treatment with remote ischemic preconditioning in pigs. Activation of STAT3 was necessary for ischemic postconditioning in pigs, while RIPC in human needs STAT5 not STAT3, this may be because the underline signaling was different from human [8][9][10][11]. Our research has also indicated that STAT3 played crucial roles in both ischemic preconditioning and postconditioning cardioprotection [12,13]. Furthermore, expression of STAT3 was found to have been reduced in the myocardium of patients afflicted with diabetes, and this may constitute the main reason why diabetes afflicted subjects were more likely to experience myocardial I/RI and were less responsive to protective treatments including pre-and postconditioning [13,14]. However, whether or not mTOR functions as an upstream effector of STAT3 in cases of diabetic myocardial I/RI is unclear. Therefore, we sought to use streptozotocin (STZ) -induced diabetic rats and high glucose exposed H9C2 cells to facilitate investigations of the roles of mTOR-STAT3 in myocardial I/RI in diabetic conditions. Our main goal was to provide evidence facilitating the identification of novel treatment targets for myocardial I/RI in diabetes.

Animals and diabetes model
All animal based experimental were examined and given approval by Institutional Committee for Animal Care and Animal Use of Wenzhou Medical University. We purchased 6-8-week-old specific pathogen free (SPF) male Sprague-Dawley rats (260 ± 10 g). We induced diabetes in rats by injection of streptozotocin (STZ; 60 mg/kg, STZ was dissolved in 0.1 M citrate buffer and pH = 4.5; Sigma, St. Louis, MO) into the tail vein and following the protocol as described in more detail previously [15]. Access to food and water for experimental animals was provided ad libitum. Conditions in the room where animals were maintained at a constant air temperature of 23 °C, a constant humidity = 50 %, and a 12hour (h) light and 12 h dark cycle.
We established I/R by ligating left anterior descending (LAD) arteries for 30 minutes (min) followed by 2 hours (h) reperfusion in the I/R afflicted groups as described before [16,17]. TTC (1%, 2,3,5-triphenyltetrazolium chloride) based staining was used to facilitate the determinations of myocardial infarction as described [10]. Upon the completion of reperfusion, normal regions of left ventricles (LV) were defined by way of ligating the left anterior descending and infusing 5 % Evans Blue (Sigma, St Louis, MO) through the right jugular vein. We then applied an overdose of pentobarbital through injection to euthanize rats. We subsequently dissected and froze hearts for 15 min at -20 °C, and then sliced the hearts into five 1-

Dihydroethidium (DHE) staining
DHE staining was used for reactive oxygen species (ROS) detection following manufacturer's protocols. Red fluorescence was emitted when DHE was oxidized by superoxide, and it was detected by fluorescence microscopy (Olympus, Tokyo, Japan).

Lactate dehydrogenase (LDH) activity
LDH is a major indicator of myocardial I/RI injury. Thus, we examined levels of LDH in H9C2 culture mediums by LDH Cytotoxicity Assay Kits (Roche, USA) followed the manufacturer's protocols.

Western blotting assays
In our animal-based experiments, we homogenized rats' LV tissues in 1× lysis buffer acquired via Cell Signaling Technology (Beverly, MA) and centrifuged samples at 13, 200 g for 30 min, while H9C2 cells in ice-cold 1× lysis buffer were centrifuged for 10 min. The supernatant was collected as total protein. We then used Bradford protein assays to determinate the protein concentrations. Then, we sampled equal amounts of protein homogenates, resolved by the SDS-PAGE (7.5-12.5%).
Next, we transferred the samples to polyvinylidene nitrocellulose membranes and completed processing as described in previously [9,10]. We purchased cleaved-caspase3, mTOR, phosphorylation-mTOR (p-mTOR), p-STAT3-Ser727, Total-STAT3, GAPDH antibodies, and secondary antibody all from Cell Signaling Technology (Beverly, MA). We detected protein bands through the standard protocols for the ECL method. Densitometry for protein band assessments was completed in Image J Software (National Institutes of Health, USA). We reported data for normalized protein band density in arbitrary units.

Statistical analyses
Analyses were performed in a blinded manner. We used the mean ± the standard error of the mean (± SEM) for values in statistical analyses. We used GraphPad Prism Software (Version 6.0) to complete statistical analyses and used one-way analysis of variance (ANOVA) and Tukey's tests to facilitate determinations of statistical significance between different treatment groups. The level of significance at which the null hypothesis of no differences between treatment groups would be rejected was p< 0.05.

Results: mTOR inhibition with Rapamycin attenuated myocardial I/RI in diabetes
As Fig. 1A indicates, mTOR inhibitor rapamycin significantly reduced sizes of post-ischemic myocardial infarction (IS) (Fig. 1A; 8-week-old diabetic rats (D8w) +I/R+rapamycin vs. D8w +I/R) and significantly reduced the release of Tn-I ( Fig. 1B; D8w+I/R+rapamycin vs. D8w+I/R). The levels of cardiac mTOR protein and phosphorylated mTOR (p-mTOR) increased significantly in D8w rats as compared non-diabetic control groups (Figs. 1C and 1D). In contrast, the levels of phosphorylation of STAT3 at Ser727 (p-STAT3-Ser727) were significantly decreased and while total STAT3 did not significantly differ between D8w rats and non-diabetic

Effects of mTOR gene knockdown on hypoxia re-oxygenation(H/R) injury in H9C2 cells
Results exemplified in Fig.3A  production assessed by DHE staining (Fig. 3D).

Discussion:
The findings from our study indicated that hyperglycemia induced significant activation of cardiac mTOR and induced reductions in p-STAT3-Ser727 activation.
Further, we showed that inhibition of mTOR could reduce myocardial I/R or H/R injury under HG condition through enhancement of p-STAT3-Ser727 activation. To our knowledge, our findings are the first to have characterized the dynamics of mTOR-p-STAT3-Ser727 with respect to myocardial I/RI in hearts impacted by diabetes, this offered additional insights regarding the mechanism of diabetic myocardial I/RI. mTOR is a type of serine/threonine kinase which functions mainly as a nutrient and energy sensor， and plays essential roles in protein synthesis and autophagy, cell growth and survival [18,19]. AMPK/mTOR has been identified as a major signaling pathway in the mTOR family and regulates autophagy. However, there is researchwhich that AMPK did not directly regulate mTOR, but instead mTOR could be regulated by Akt [20]. In the current study, we found that the levels of mTOR and p-mTOR were both increased, and other research indicated that mTOR participates in the regulation of autophagy and can be regulated by other upstream effectors, including oxidative stress [21]. It has been reported that mTOR modulated autophagy and protected cardiomyocytes from oxidative stress-induced toxicity [22,23], whereby vice versa, oxidative stress may provoke mTOR activation and subsequently impair autophagy [24]. Research has also indicated that mTOR played crucial roles in the dynamics underlying the balance of cardiac systems. mTOR has been shown to be essential in preventing apoptosis during ischemic postconditioning mediated cardiac protection in rats [23]. In our study, application of mTOR inhibitor rapamycin in in vivo study or mTOR gene knockdown in H9C2 cells both decreased the myocardial I/RI or H/R injury, manifested by significantly decreased ROS level that was concomitant with reduced expression of protein cleaved-caspase3 and significant reduction in apoptotic cell death. Continuous activation of mTOR was detrimental to vascular systems [25], and thus reducing the activation of mTOR could reduce vasculopathy and improve microcirculatory coronary flows following cardiac transplantation [26,27]. Based upon these findings, mTOR not only plays a key role as a regulator of autophagy but also plays an important role with respect to myocardial I/RI through its effect upon other types of signaling. In our study, we found that reduced activation of mTOR consequently decreased post-ischemic ROS levels and oxidative damage manifested as reductions in 15-F2t-Iso and MDA. Thus, we hypothesize that inhibition of mTOR in diabetes may have conferred myocardial protective effects through reducing oxidative stress, although the underlying mechanisms and dynamics merit additional rigorous investigations.
Signal transduction and activation of transcription 3 (STAT3) as the major memb er of signaling pathway of survival activating factor enhancement pathway (SAFE) ha s significant roles in myocardial I/RI especially diabetes [12]. The expression of STA T3 decreased in the heart of diabetes which may be the reason why diabetic hearts are prone to myocardial I/RI [22]. STAT3 was essential for ischemic preconditioning (IP C), ischemic postconditioning (IPostC) and remote IPC (RIPC). However, as Heusch et al reported, in separate IPC, IPostC and RIPC studies, the STAT3 phosphorylation at-Tyr705 (p-STAT3-Tyr705) were all significantly increased by IPC, IPostC or RIPC.
However, when analyzed the phosphorylation of STAT3 and other signaling proteins in left ventricular biopsies of IPC, IPostC and RIPC in one approach, they only observ ed the STAT3 phosphorylation at-Tyr705 at early reperfusion was significantly increa sed along with infarct size reduction by IPC, only trend wise by IPostC and RIPC. Th ese studies demonstrated that STAT3 phosphorylation at-Tyr705 is necessary conditio ning cardioprotectionbut it may have its own specific time course in myocardial condi tioning [2,9,10,16]. Janus kinase 2(JAK2) is one of the upstream effectors of STAT3 production, and as reported STAT3 Tyr705 was directly regulated by JAK2 whereas STAT3 Ser727 was regulated by ERK/MAPK [23]. Signal-transducing protein kinase s are the mitogen-activated protein kinases (MAPK) that could be activated in cardiac cells under stress, it can phosphorylate various substrates including transcription facto rs such as abovementioned STAT3. MAPK activation needs both tyrosine and threon ine phosphorylation, and MAPK may be upstream of ERK, JNK and p38 in myocardi al ischemia, reperfusion and ischemic preconditioningas well as in other pathophysiol ogical conditions [28]. Some researchers have reported that STAT3 was also affected b y protein kinase C (PKC) [13,29], while other findings have provided evidence that S TAT3 has effect on autophagy by way of serving as the downstream effector of mTO R [30]. In this study, we found that mTOR gene knockdown could subsequently restor e I/R or H/R induced reduction in p-STAT3 Ser727 and attenuate myocardial I/RI. Re cent research appears to support our findings in that treatments with rapamycin increa sed the levels of expression of p-STAT3-Tyr705 [31]. Thus, our findings suggested th at mTOR is an important regulator in myocardial I/RI. However, if mTOR regulates a utophagy through STAT3, the dynamics and mechanism underlying this process need further research. Autophagy can have protective effects during ischemia while contra stingly plays detrimental roles during reperfusion, thus, the relationship between STA T3 and autophagy deserves more research [32,33]. Next, we may need to do an in-dep th study using the inhibitor of STAT3 or cardiac specific STAT3 knock out to observe the role of STAT3 in diabetic myocardial I/RI. In our another study exploring the effe cts of remote ischemia preconditioning (RIPC), we demonstrated that STAT3 gene kn ockdown cancelled RIPC mediated cardioprotective effect, and that RIPC conferred it s cardioprotection against myocardial I/RI mainly through PKC-STAT3(Ser727) [13], in this study, we also did not observed the changes of p-STAT3-Tyr705. While, in th e study of Heusch et al' they found that phosphorylation of STAT3 at Tyr705 was ess ential in ischemia preconditioning, ischemia postconditioning and RIPC. This differen ce may be due to that we focused on studying different the upstream regulator of STA T3 and that the animal models used were also different. In the current study, we focus ed on exploring the role of mTOR in diabetic myocardial I/RI. In the following study, we shall do more research to define the roles and interplays in between mTOR and ST AT3 and their impact on autophagy in the context of myocardial I/RI in diabetes.
Our study has the limitation that we just used the type 1 diabetes model, while in clinical settings most diabetic patients were type 2 diabetes. Compared to type 2 diabetes, the major pathological change of type 1 diabetes is the insulin deficiency and high glucose was the primary element. The type 2 diabetes was a polygenetic disease, it can be divided into two kinds, with or without obesity, and several genes can predispose individuals to developing the disease, so its mechanism is more diverse. Therefore, on the basis of our initial findings regarding the mechanism of myocardial I/RI in type 1 diabetes, future study shall be extended to study the impacts and mechanisms of type2 diabetes on myocardial I/RI.

Conclusion:
Our study demonstrated that increased activation of mTOR in the heart of type 1 diabetic rats causes subsequent reduction in p-STAT3-Ser727 and exacerbated myocardial I/RI. Further, we found that knockdown of mTOR can increase the expression p-STAT3-Ser727 and reduce cardiomyocyte H/R injury. The mechanistic insights gained in the current study may help facilitate the development of targeted therapies against myocardial I/RI in diabetes.