Nogo-A mediated endoplasmic reticulum stress during myocardial ischemic-reperfusion injury in diabetic rats

Background: Diabetic myocardial ischemia reperfusion (MI/R) injury is aggravated after myocardial infarction, which leads to myocardial damage. Molecular mechanisms associated with the diabetic ischemia-related cardiac diseases are not yet fully understood. Nogo-A is an endoplasmic reticulum (ER) protein ubiquitously expressed in tissues including in the heart. However, the mechanisms that account for the Nogo-A in MI/R injury remain unknown. Methods: SD (Sprague Dawley) rats were subjected to 45 min of ischemia, followed by 3 h reperfusion. Rats were injection with streptozotocin (60mg/kg), tauroursodeoxycholic acid injection (100mg/kg) or corresponding controls just prior to MI/R. Blood and heart samples were collected at 3 h post-reperfusion. Serum LDH and CK-MB, myocardial infarct size, histopathologic changes, apotosis and ER stress were analyzed to evaluate MI/R injury. Signaling pathways were also investigated in vitro using embryonic rat cardiomyocyte-derived H9c2 cells cultures to identify underlying mechanisms for Nogo-A in diabetic MI/R injury. Results: TUDCA treatment significantly reduced Nogo-A, GRP78 and CHOP levels, diminished myocardial infarct areas, attenuated ER stress and decreased myocardial apoptosis after MI/R. ER stress signaling was significantly decreased in the TUDCA-treated MI/R group compared with controls. The effect of Nogo-A was abrogated by pretreatment with knockdown CHOP. A positive feedback loop between Nogo-A and CHOP was found leading to an enhanced ER stress in diabetic MI/R injure. Conclusions: Our data suggest that Nogo-A mediated ER stress plays a major role in diabetic MI/R injury and Nogo-A might be a key regulator of ER stress.


Background
Diabetes mellitus (DM) is one of the leading causes of morbidity and mortality in afflicted individuals. The International Diabetes Federation data have shown that there are more than 463 million people with DM worldwide, and it could rise to over 700 million or more by 2045 (Saeedi, et al. 2019). Moreover, DM is an important risk factor for cardiovascular disease. The incidence rate of ischemic cardiomyopathy in diabetic patients is higher than non-diabetic patients (Russo, et al. 2017). Previous studies have shown that diabetic patients suffering from myocardial ischemia reperfusion injury leads to more serious impairment of heart function, higher incidence of myocardial infarction and heart failure . Mechanisms contributing to the pathogenesis of diabetic MI/R injury are multifactorial and complex but also highly integrated. Many studies have shown that ER stress played important role in the pathogenesis of diabetes, and suppressed ER stress contributed to reduced cardiac infarct size in type 2 diabetes (Arunagiri, et al. 2018;Mali, et al. 2018).
The ER stress is a complex membranous network found in all cells where it plays an important role in calcium homeostasis, proteins folding, and lipid biosynthesis (Dominguez-Martin, et al. 2018;Marchi, et al. 2018;Ruan, et al. 2020). Clinical studies have shown that heart expression of ER stress markers with established diabetic MI/R in patients is higher than patients with non-diabetic, suggesting a heightened ER stress response in the heart of diabetic MI/R injury patients (Guo, et al. 2017). A wide variety of stressors, including oxidative stress and ischemia, disrupt endoplasmic reticulum function, which leads to protein misfolding and unfolded protein response (UPR) (Hong, et al. 2017;Jin, et al. 2017). Cumulating evidence suggests that C/EBP homologous protein (CHOP) is involved in the pathogenesis of diabetes, in response to glucotoxicity, lipotoxicity, as well as oxidative stress and islet amyloid derived from IAPP (Yang, et al. 2017). Diabetic CHOP knockout mice seemed to be protected from myocardial ischemia reperfusion, CHOP has been implicated in exaggerated reactive oxygen species (ROS) production by upregulation of the UPR-regulated oxidative protein folding machinery in the ER, which is directly contributing to ROS generation through the oxidation of disulfide bonds (Nam, et al. 2015). However, the mechanism of CHOP mediated ER stress in diabetic MI/R injury remains largely unknown.
Neurite outgrowth inhibitor proteins (Nogo) belong to reticulon protein family, which is characterized by the ER targeting motif at the carboxy terminal (Long, et al. 2017). Nogo gene encodes three splicing isoforms, Nogo-A, Nogo-B and Nogo-C, they lack an N-terminal signal sequence and are predominantly localized to the ER (Mohammed, et al. 2020). Nogo-A is the longest protein in Nogo family, and expressed in many tissues, including heart (Sarkey, et al. 2011). Nogo-A is well characterized as a potent inhibitor of axonal regeneration and plasticity in the central nervous system, however, the role of Nogo-A in non-nervous tissues is essentially unknown. A previous study has shown that Nogo-A expression was shown to be significantly increased in plasma from patients who have experienced a coronary heart disease is associated with increase reactive oxygen species levels and promote apoptosis (Ding, et al. 2017). However, Nogo-A is expressed in cardiomyocytes whether related to ER stress and its precise functions in diabetic ischemia cardiomyopathy remain poorly understood. Accordingly, the aims of the present study were to identify the role of Nogo-A during MI/R in diabetic hearts and examine a key role of Nogo-A in mediated endoplasmic reticulum stress.

Animals
Sprague-Dawley rats (2-month-old, 200-220 g) were obtained from the Laboratory Animal Services Centre of Wuhan University. During the study, the animals were housed in an animal room (temperature: 20 ± 2°C, humidity: 60% ± 5%, 12-hour light/dark cycle). With free access to standard rat chow and tap water. All institutional and national guidelines for the care and use of laboratory animals were followed and approved by the appropriate institutional committees.

Induction of diabetes
Diabetes model was induced by a single left intraperitoneal injection with streptozotocin (STZ, Sigma, USA) 60 mg/kg, whereas normal control rats were injected with the same volume of mother solution. Three days after the STZ administration, fasting blood glucose continued to be higher than or equal to 16.7 mmol/L, the diabetes model was successfully prepared. All invasive operations in our experiment were performed under anesthesia with 3% pentobarbital sodium (50 mg/kg) (Sigma-Aldrich, USA). The TUDCA group was intraperitoneally injected with TUDCA (100 mg/kg) or equal volumes of mother solution in control groups. During the experiment, the dosage was adjusted according to the body weight of the rats, the indicators were monitored regularly.

Myocardial ischemia and reperfusion
After 8 weeks diabetes induction, the diabetic animals were anesthetized with pentobarbital sodium (50 mg/kg), placed face up and fixed on the operating table, and connected to electrocardiogram wires. After tracheal intubation and connection to a small animal respirator (tidal volume of 8-12 mL, 1:2 breathing, respiratory rate 70-80 times/min), the chest was opened between the left third and fourth rib exposing the heart. A 7-0 nylon suture was placed around the left anterior descending coronary artery. All groups were made ischemic for 45 minutes by ligating the artery, and this was followed by reperfusion for 180 minutes by loosening the ligature. Arrhythmias were monitored during ischemia-reperfusion by electrocardiogram. ST-segment elevation and widening of R wave indicated ischemia. A 50% drop-off in ST-segment elevation was indicative of successful reperfusion.

Cell culture
The H9c2 cells were cultured in DMEM containing 10% FBS in a humidified incubator with 5% CO2 in air at 37 °C. For albumin overloading experiments, H9c2 cells were cultured with Endotoxin-free human serum albumin (Sigma-Aldrich Co., Ltd, St. Louis, MO, USA).

High glucose procedure and hypoxia-reoxygenation injury
H9c2 cells were subjected 50% glucose (30 mM) for the HG procedure. Culture dishes were placed inside humidified incubator with 5% CO2 in air at 37 °C for 24 h, unless noted otherwise. Following, the H/R procedure was performed. For hypoxia exposure, cells were maintained under anoxic conditions in chambers gassed with a mixture of 95% N2, 5% CO2, and 1% O2 at 37°C for 4 h. For reoxygenation, plates were removed from the anoxic chamber to a normoxic chamber for 2 h.

Cell Transfection
The shRNA targeting CHOP (GenePharma Co., Ltd, Shanghai, China) or the negative shRNA were transiently transfected into cells using Attractene transfection reagent (Qiagen Co., Ltd, Hilden, Germany) according to the manufacturer's instructions.

Quantitative Reverse Transcription PCR
Total RNA extracted by Trizol (ThermoFisher Co., Ltd, Shanghai, China) and PureLink RNA Mini Kit (ThermoFisher Co., Ltd, Shanghai, China) was reverse-transcribed using TIANScript II RT Kit (Tiangen Biotech Co. Ltd., Beijing, China) following the instructions of the manufacturers. qRT-PCR was carried out using RealMasterMix (SYBR Green, Tiangen Biotech Co. Ltd., Beijing, China). Three parallel wells were set for each group. The relative quantifications of Nogo-A and CHOP expressions were normalized to GAPDH. The primers used in this study are listed in Table 1. The GAPDH was used as the internal reference, and the 2 -ΔΔCt method was used for transcriptional change evaluation. The formula was expressed as ΔΔCt = ΔCt experimental group-ΔCt control group in which ΔCt = Ct target gene-Ct internal reference. Table 1 Sequences of primers used in qRT-PCR

Myocardial infarct size
To evaluate the size of the infarct area (IA), risk area (AAR) and left ventricle (LV), six rats in each group. The left femoral vein of the rat was separated, and 2 ml of 2% Evans Blue was injected into the heart through the left femoral vein immediately after 3 hours of reperfusion. Animals were sacrificed, the heart was taken out and washed with phosphate buffer saline and then placed in a -70 °C for 15 minutes. Hearts were sliced into 2 mm thick sections and incubated with freshly prepared 1% TTC solution at 37 °C for 15 min. TTC stained the viable part red, and the infract part remained pale. The infarct size was analyzed by the image analysis system (Image-J). The percentage of area at risk versus left ventricle (AAR/LV × 100%) and infarct area versus area at risk (IA/AAR × 100%) were calculated.

Cell Viability
Cell viability was determined by cell counting kit-8 (Dojindo, Kumamoto, Japan) in 96-well plates according to manufacturers' instructions. In brief, the CCK-8 solution was added to each well after the treatments and incubated for 3 h. The absorbance at 450 nm was measured using a microplate reader. The mean optical density of 6 wells in each group was used to calculate the percentage of cell viability.

Immunohistochemical staining
Immunohistochemical staining was performed according to the manufacturer's instruction. Briefly, the deparaffinized and rehydrated specimens were incubated for 2 h at 37 °C with monoclonal antibody, and then washed and incubated with HRP goat anti rabbit IgG antibody for 30 min. The sections were stained with DAB solution for 1 min and the nucleus was counterstained with hematoxylin.

Apoptosis
In vivo study: TUNEL assay (Beyotime, shanghai, China) was performed with a commercially available kit. Apoptotic cardiomyocytes were subjected to TUNEL staining. In vitro study: after H/R, cells were collected and resuspended in binding buffer and incubated with fluorescein isothiocyanate conjugated annexin V and propidium Iodide for 15 minutes in the dark. All manipulation strictly followed the manufacturer's instructions. Cellular fluorescence was measured using a FACS Calibur flow cytometry (BD Biosciences, USA). The data obtained from the cell population were analyzed using Cell Quest Pro software (BD Biosciences, USA).

Western blot
Tissue or cells were lysed with RIPA buffer containing protease and phosphatase inhibitor cocktail. After quantifying protein concentration, lysates were subjected to western blot analysis using specific antibodies against Nogo-A (Santa Cruz Biotechnology, Santa Cruz, CA), cleaved caspase-3(Cell Signaling Technology, MA, USA), CHOP (Cell Signaling Technology, MA, USA), GRP78(Cell Signaling Technology, MA, USA), GAPDH (Cell Signaling Technology, MA, USA). We repeated each Western blot analysis using protein from three different and separate experiments. The specific protein bands were analyzed using Odyssey Application Software 3.0 to obtain the integrated intensities, followed by linear regression of the intensity data.

Statistical analysis
Data are shown as the mean ± SEM. Comparisons between multiple groups were made by one-way ANOVA followed by the Tukey test. GraphPad Prism 6 software was used for statistical analyses. P values < 0.05 were considered statistically significant.

Body weight change and blood glucose level
After 8 weeks of STZ-induced diabetes, the rats showed characteristic symptoms of type 1 diabetes including polydipsia and polyphagia. In all experimental groups, body weight was measured at 1st, 2th, 4th and 8th week after STZ injection (Table 2). Fasting blood glucose level in all experimental groups was measured at 1st, 2th, 4th and 8th week (Table 3). Bodyweight was lower, whereas the blood glucose level was higher than that in the agematched control rats.

Increased MI/R injury in diabetic rats compared with non-diabetic rats
To investigate the effects of diabetes status on MI/R injury, we next measured myocardical infarct size and serum biochemical markers in each experimental group. Compared with the NS group, serum LDH (Fig. 1A) as well as CK-MB (Fig. 1B) levels were significantly increased in the DS group at baseline. Diabetic rats subjected to MI/R showed larger infarct sizes (Fig. 1C), higher serum LDH and CK-MB levels. These results show that diabetic hearts are more vulnerable to MI/R injury. All the results are presented as mean ± SEM, n=6/group. * p < 0.05 versus NS group, # p < 0.05 versus NIR group, and ▲ p < 0.05 versus DS group.

The ER stress response occur concomitantly with Nogo-A upregulation in diabetes
To understand the pathological role of Nogo-A in the diabetic MI/R injury heart, we checked the Nogo-A expression level in diabetic rats. In vivo, immunohistochemical staining by Nogo-A, GRP78 and CHOP antibody showed that Nogo-A, GRP78 and CHOP were upregulated in the DIR group ( Fig. 2A-D). Western blot result confirmed the increased Nogo-A, GRP78 and CHOP proteins level in the DIR group (Fig. 2E-H). Together, these results showed that in diabetic rats Nogo-A and ER stress markers proteins were changed during MI/R injury, suggesting that Nogo-A and ER stress may have the role in the pathogenesis of ischemia-related cardiac diseases in diabetes.

Figure 2
Expression of Nogo-A and ER stress markers was increased in the diabetes MI/R injury. (A) Immunohistochemistry staining for Nogo-A and ER stress markers in MI/R injury heart and the representative pictures are shown (bars = 50 um). (B) Semi-quantitative data of Nogo-A, GRP78 and CHOP staining in different groups of rats. (C) The heart tissue lysates were subjected to western blot analysis with specific antibodies against Nogo-A, GRP78 and CHOP. (D)The densitometry analyses of western blots are shown. Values are expressed as means ± SEM. n=6/group. * p < 0.05 versus NS group, # p < 0.05 versus NIR group, and ▲ p < 0.05 versus DS group.

Accelerated Nogo-A and MI/R injury in diabetic rats was attenuated by TUDCA treatment
Three weeks after STZ administration, we intraperitoneally injected diabetic rats with TUDCA (100 mg/kg), an ER stress inhibitor, or equal volumes of saline twice daily for five weeks. We found that the diabetic rats treated with TUDCA had greatly reduced Nogo-A levels after MI/R (Fig 3A) as assessed by western blot of Nogo-A. The ER markers (GRP78, CHOP) of TUDCA treated rat hearts also decreased as comparing with DIR group after MI/R ( Figure 3B-C) as assessed by western blot. In addition, we investigated the MI/R-induced cardiomyocytes apoptosis in diabetic rats and found that was alleviated by TUDCA treatment as assessed by the measurement of cleaved caspase-3 ( Fig. 3D-E) and Tunel assay (Fig. 3F).
Suggesting that inhibition of ER stress by TUDCA, a pharmacological inhibitor of ER stress, reduce Nogo-A expression levels and attenuated MI/R injury in diabetic rats from ER stress.

Figure 3
Expression of Nogo-A and ER stress markers was decreased in the diabetic rats treated by TUDCA. D: STZ-induced diabetic rats; S: sham operation; IR: ischemia/reperfusion; DIR group rats was subjected to 45 min of ischemia, followed by 3 h of reperfusion, TUDCA: (250 mg/kg) twice daily for five weeks. (A) The heart tissue lysates were subjected to western blot analysis with specific antibodies against Nogo-A. (B) The heart tissue lysates were subjected to western blot analysis with specific antibodies against GRP78. (C) The heart tissue lysates were subjected to western blot analysis with specific antibodies against CHOP. (D) TUNEL assay was performed with a commercially available kit. Apoptotic cardiomyocytes were subjected to TUNEL staining (bars = 100 um). (E) Quantitative analysis of TUNEL staining in heart sections.

H/R injury of H9c2 cells aggravated under high glucose conditions
To verify the in vitro model of MI/R injury, we performed in vitro study in cultured embryonic rat cardiomyocyte-derived H9c2 cells. We measured cells viability, LDH release in the experimental groups. We found that HGH/R group resulted in a sharp increase in LDH (Fig. 4A). Furthermore, as shown in (Fig. 4B), H9c2 cells viability was significantly lower in the HGH/R group than the LG group. These results suggested that increased susceptible of H9c2 cells in high glucose.

Figure 4
Effects of H/R on cell viability, LDH release in H9c2 cells. LG: low glucose; HG: high glucose (30 mM); HR: hypoxia/reoxygenation; H9c2 cells were exposed to hypoxia for 4 h and reoxygenated for 2 h under LG or HG stimulation. (A) LDH release in the supernatants was measured using a kit, (B)Cell viability was detected using an MTT assay. Data are expressed as means ± SEM; n=6/group. * p < 0.05 versus LG-N group, # p < 0.05 versus LG-HR group, and ▲ p < 0.05 versus HG-N group.

knockdown of Nogo-A attenuated ER stress in H9c2 cells exposed to high glucose
As Nogo-A belongs to the RTNs family, which is especially enriched in the ER, we wondered whether Nogo-A knockdown could relieve albumin-induced ER stress and apoptosis in H9c2 cells in diabetic MI/R injury. The qRT-PCR results showed that contrasted with the sh-scramble group, the expression level of Nogo-A was significantly decreased after sh-Nogo-A transfection, indicating successful transfection (Fig. 5A). We found that knockdown of Nogo-A attenuated HSA-induced ER stress under high glucose H/R group, as reflected by decreased expression of GRP78 and CHOP (Fig. 4A), indicating that H9c2 cells ER stress was activated in response to HSA stimulation and was alleviated by knockdown of Nogo-A. In addition, we investigated the role of Nogo-A in albumin-induced H9c2 cells apoptosis and found that knockdown of Nogo-A cleaved caspase-3 (Fig. 4E) and FACS Calibur flow cytometry (Fig. 4G). Collectively, these data indicate that Nogo-A contributes to protein overloading induced ER stress and plays a critical role in H9c2 cells injury and apoptosis under HGH/R. Values are expressed as means ± SEM. * P < 0.05 compared to scramble, ** P < 0.01 compared to scramble; # P < 0.05 compared to shNogo-A; ▲ P < 0.05 compared to H/R+scramble, n = 6.

CHOP positively regulated Nogo-A expression during MI/R in diabetes
Finally, to understand how Nogo-A contributes to ER stress and apoptosis in diabetic MI/R injury, we examined the interaction between Nogo-A and CHOP, a key ER stress transcriptional factor leading to the activation of apoptosis. The qRT-PCR results showed that contrasted with the sh-scramble group, the expression level of CHOP was significantly decreased after sh-CHOP transfection, indicating successful transfection (Fig. 6A). In the previous experiments we have found that CHOP was extremely increased in diabetic MI/R both in vivo and vitro. We knocked down CHOP specific shRNA in H9c2 cells, then stimulated with HSA for 48 h and exposed to high glucose for 24 h, followed by 4 h of hypoxia and 2 h of reoxygenation showed that knockdown of CHOP significantly suppressed Nogo-A expression (Fig. 5A-D), indicating a positive feedback loop between CHOP and Nogo-A. In addition, CHOP depletion also protected cardiomyocytes from HGH/R-induced apoptosis assessed by flow cytometry analysis using FACS Calibur flow cytometry labeling ( Fig. 5E-F). Taken together, our data suggest that CHOP has a positive feedback to Nogo-A and down-regulation CHOP can protect diabetic heart from apoptosis. SEM. * P < 0.05 compared to scramble, ** P < 0.01 compared to scramble; # P < 0.05 compared to shCHOP; ▲ P < 0.05 compared to H/R + scramble, n = 6.

Discussion
Nogo family proteins are profoundly involved in multiple cellular processes especially the morphology and functional science of ER. Our present study found that Nogo-A is a determinant player in ER stress and mediated cardiomyocyte apoptosis during MI/R in diabetes. There are several lines supporting our notions. First, Nogo-A protein is increased in diabetic MI/R heart and also increased in H/R stimuli-induced cardiomyocytes under high glucose. Second, accelerated Nogo-A and MI/R injury in diabetic rats was attenuated by TUDCA treatment and knockdown of Nogo-A per se is sufficient to decrease ER markers as well as prevents cardiomyocyte apoptosis. Third, we identified CHOP is a target gene of Nogo-A, in vitro knockdown of CHOP protected cardiomyocytes against apoptosis after H/R under high glucose, which positively regulated Nogo-A and is upregulated during MI/R in diabetes.
While timely reperfusion has proved to be an invaluable tool, MI/R injury represents a mechanism that may limit its effectiveness. We and others have previously shown that the diabetic myocardium is more susceptible to ischemia-reperfusion injury, possible causes are increased oxidative stress, inflammation response, apoptosis, ER stress or mitochondrial dysfunction, but further mechanisms are still to be studied (Leng, et al. 2018;Samidurai, et al. 2020;Qiu, et al. 2021). ER stress is an important feature leading to the cardiac dysfunction after ischemic heart diseases in diabetes. The activation of the mild stress appears to have a cardioprotective role, ER-associated degradation is activated by UPR to clear irreparably misfolded proteins. However, when severely stress the UPR fails to reduce ER stress and restore homeostasis, ER stress causes cell dysfunction and apoptosis. Thus, the environment in the ER must be optimal for efficient synthesis and folding of these important proteins. In this setting, ER stress lead to cardiovascular complications. It has been suggested that Nogo serves an important role in ER homeostasis may contribute to myocardial I/R damage (Weng, et al. 2018;Samidurai, et al. 2020). In this study we found that both Nogo-A protein level and ER stress markers response were increased in diabetic MI/R injury heart, and treat with TUDCA can protect diabetic MI/R heart. There are many possible causes of a relationship between ER stress and diabetic myocardial vulnerability increases. Other studies have shown that diabetes-induced ER dysfunction through neuregulin-1 and O-linked beta-Nacetylglucosamine (Ngoh, et al. 2009;Fang, et al. 2017). Other study shows that hyperglycemia induced ER stress in rats and significantly lowered the expression of glucose transporter proteins, misfolded insulin was shown to cause diabetes in both mouse models and humans (Lakshmanan, et al. 2013). Recent studies indicated that in perioperative diallyl trisulfide treatment effectively ameliorates MI/R injury in type 1 diabetic setting by suppressed PERK/eIF2α/ATF4/CHOP-mediated ER stress level, thus reducing myocardial apoptosis and eventually preserving cardiac function (Yu, et al. 2017). This is consistent with our recent in vivo studies showing that ER stress markers increase and heart injury aggravation in diabetic rats during MI/R. At present, there is no satisfactory way to cure or mitigate diabetic MI/R injury, hence, a deeper understanding of the underlying molecular mechanisms of this disease is essential to the development of new effective therapies.
Here, we reported a novel ER stress marker, Nogo-A, which was highly expressed in diabetic ischemic heart and suppressed by CHOP, a key marker of ER stress. Nogo-A is an important neurite growth-regulatory protein in the adult and developing nervous system, and are involved in neuroendocrine secretion or membrane trafficking and apoptotic processes (Farrer and Kartje 2018). Our above results on Nogo-A and others' previous studies on Nogo-B or Nogo-C indicate that the increased Nogo proteins during ischemic heart diseases may contribute to cardiac dysfunction through regulating cardiomyocyte apoptosis. To better understand the pathophysiological significance of Nogo-A in the diabetic heart, we generated the Nogo-A knockout model in vitro. Our in vivo functional study Nogo-A in rats provides solid evidence supporting our hypothesis, that Nogo-A is extremely increased after MI/R in diabetic rats. Then we found that depletion of Nogo-A protected cardiomyocyte apoptosis in HGH/R H9c2 cells, largely decreased HGH/R injury, and most importantly, decreased ER stress after H/R, suggesting that Nogo-A may serve as a target for ER stress, and can treatment of ischemia-related cardiac diseases. In addition, Nogo-A expression is increased in human dilated cardiomyopathy and ischemic hearts as well as involved in glucose homeostasis (Sarkey, et al. 2011;Bonal, et al. 2013;Ding, et al. 2017). It was also stated that Nogo-A knockdown inhibits H/R-induced activation of mitochondrial-dependent apoptosis in cardiomyocytes (Sarkey, et al. 2011), unlike our study, they showed that knockdown of Nogo-A inhibited H/R-induced cleaved caspase-3 cleavage without affecting ER stress. This different in high glucose model may account for the different results. Our present study of the protective effect of Nogo-A knockout in the H9c2 cells is in general agreement with Nogo family protein Nogo-C knockout mouse model (Jia, et al. 2016). Although Nogo-A knockout protected the cardiomyocytes from H/R damage under high glucose, it showed no cardiac phenotype at basal level, suggesting that Nogo-A is either dispensable for normal functions or there are redundant pathways in the heart. These possibilities should be examined to further clarify the role of Nogo-A.
How Nogo-A mediates ER stress remains unclear. The findings from this study expand our understanding of the regulatory role of ER stress in diabetic MI/R induced apotosis. We focused on the regulation of Nogo-A by CHOP, given that excessive CHOP is linked to maladaptive ER stress. We have shown that knockdown of Nogo-A prevented H/R induced activation of CHOP under high glucose. It is known that CHOP deletion can protect cardiomyocytes from apoptosis through inhibition PERK/CHOP pathway (Nam, et al. 2015). In vivo, CHOP deficiency attenuated apoptosis, inflammation, fibrosis in fat-induced liver injury (Toriguchi, et al. 2014), targeted disruption of the CHOP gene significantly prevented Ins2C96Y-induced diabetes by decreasing ER stress-mediated apoptosis in β cells (Oyadomari, et al. 2002). These data suggest a critical role of CHOP in disease development. Here, we found that knockdown of CHOP suppressed Nogo-A expression and Nogo-A-mediated ER stress and apoptosis in diabetic MI/R injury, suggesting Nogo-A expression is regulated by CHOP. These data together suggest a mechanism that Nogo-A induces CHOP expression, forming a positive feedback manner to synergistically stimulate ER stress in diabetic heart.
There are some limitations to our study. First, although we found abnormal increase in Nogo-A in diabetic MI/R injury in both in vivo and in vitro models, we only knocked down Nogo-A in vitro, and further research is needed to Nogo-A gene knockout on mice explore its deep mechanism. Second, we have only made a mechanism on the cell line, and further research requires use of primary cultures of neonatal rat cardiomyocytes. However, this study at least demonstrates the feasibility of conducting such studies on mammalian cardiomyocytes, and uses these findings to further understand the relationship between Nogo-A and ER stress signaling pathways in diabetic cardiomyocytes and provided a theoretical and experimental basis to put these findings into further understanding of the mechanisms in the myocardial systems under both physiological and pathological conditions.

Conclusions
In summary, this study demonstrated that Nogo-A expression significantly increased in diabetic MI/R injury, and knockdown of Nogo-A using shRNA can be relieved ER stress and apoptosis. Furthermore, CHOP has a positive feedback to Nogo-A and down-regulation CHOP can protect diabetic heart from apoptosis. Our findings provide a novel mechanism involved in the pathophysiology of MI/R injury in diabetes. To promoting the prevention and treatment of diabetic MI/R injury, more comprehensive work on Nogo-A need to be conducted in the future.

Sources of Funding
This work was supported by grants from the National Natural Science Foundation of China (81970722, 81901947) and Hubei Provincial Natural Science Foundation (2017CFB460).

Ethical Approval
No human studies were carried out by the authors for this article.