Oncostatin M (OSM) protects against cardiac ischaemia/reperfusion injury in diabetic mice by regulating apoptosis, mitochondrial biogenesis and insulin sensitivity

Oncostatin M (OSM) exhibits many unique biological activities by activating Oβ receptor. However, its role in myocardial I/R injury in diabetic mice remains unknown. The involvement of OSM was assessed in diabetic mice which underwent myocardial I/R injury by OSM treatment or genetic deficiency of OSM receptor Oβ. Its mechanism on cardiomyocyte apoptosis, mitochondrial biogenesis and insulin sensitivity were further studied. OSM alleviated cardiac I/R injury by inhibiting cardiomyocyte apoptosis through inhibition of inositol pyrophosphate 7 (IP7) production, thus activating PI3K/Akt/BAD pathway, decreasing Bax expression while up-regulating Bcl-2 expression and decreasing the ratio of Bax to Bcl-2 in db/db mice. OSM enhanced mitochondrial biogenesis and mitochondrial function in db/db mice subjected to cardiac I/R injury. On the contrary, OSM receptor Oβ knockout exacerbated cardiac I/R injury, increased IP7 production, enhanced cardiomyocyte apoptosis, impaired mitochondrial biogenesis, glucose homoeostasis and insulin sensitivity in cardiac I/R injured diabetic mice. Inhibition of IP7 production by TNP (IP6K inhibitor) exerted similar effects of OSM. The mechanism of OSM on cardiac I/R injury in diabetic mice is partly associated with IP7/Akt and adenine mononucleotide protein kinase/PGC-1α pathway. OSM protects against cardiac I/R Injury by regulating apoptosis, insulin sensitivity and mitochondrial biogenesis in diabetic mice through inhibition of IP7 production.


Introduction
Cardiovascular complications are responsible for the leading cause of death among patients with diabetes [1]. Diabetes is now considered to be a risk equivalent of coronary artery disease for future MI and cardiovascular death [2]. The presence of diabetes has a negative impact on the outcome of the patients with acute coronary syndromes [3]. As a result of the exposure to abnormal substrate and cytokines, our previous studies have demonstrated that the myocardium of diabetic patients is more vulnerable to cardiac I/R injury than those individuals without diabetes [4,5]. However, the effective strategies which can reduce cardiac I/R injury in diabetic conditions are not well developed.
Mitochondria play a key role in diabetes and cardiac I/R injury by regulating energy homoeostasis and is emerging as a key target in cardiometabolic disease therapy [6]. Cardiac function maintenance is critically dependent on mitochondrial oxidative phosphorylation as a major source of adenosine triphosphate (ATP) [7]. Cardiac mitochondria represent a key actor of the biological systems addressed to prevent any mismatch between ATP production and utilization, and thus any major disruption in the energy available for cardiac function [8]. Abundant evidence have suggested that mitochondrial dysfunction is also a main cause of insulin resistance, one of the main characteristics of type 2 diabetes, and diabetes related cardiac comorbidities [9]. Factors which contribute to mitochondrial dysfunction, such as mitochondrial biogenesis and oxidative stress, can also lead to insulin resistance in different insulin-target tissues, and its association with mitochondrial dysfunction can culminate in the development of cardiovascular diseases [6]. A growing number of studies confirm that modulating mitochondrial survival/cell-death pathways will subsequently affect cardiomyocyte apoptosis and necrosis [7]. In this regard, therapies that enhance mitochondrial biogenesis may increase insulin sensitivity as well as decrease cardiomyocyte apoptosis and necrosis during I/R injury in diabetic mice.
Oncostatin M (OSM), produced by activated T lymphocytes, monocytes and macrophages, is an inflammatory cytokine that belongs to the interleukin-6 (IL-6) class of cytokines [10]. OSM exerts a variety of physiological and pathophysiological functions such as inflammation, tissue remodelling and cell growth [11]. OSM induces stromal cell derived factor 1 and VEGF in cardiomyocytes, modulates extracellular matrix degradation and is also involved in the modulation of smooth muscle cell proliferation [12][13][14]. Kubin et al. [15] demonstrated that inhibition of OSM signalling suppressed cardiomyocyte remodelling, resulting in deterioration of heart function after MI. Inositol pyrophosphate 7 (IP7), formed by a family of IP6Ks, represents a physiological inhibitor of Akt which mediates survival signal [16]. Our previous study indicated that Akt signalling was impaired in diabetic mice underwent cardiac I/R injury [17]. The effects of Akt inhibition by IP7 was also approved in our previous study [18]. However, no efforts have been made to investigate (i) whether OSM protects against cardiac I/R injury in diabetic mice and (ii) the underlying mechanisms responsible for OSM in modulating myocardial I/R injury in diabetic mice.

Animals
The experiments were performed in adherence with the National Institutes of Health Guidelines on the Use of Laboratory Animals and were approved by the Fourth Military Medical University Ethic Committee on Animal Care (Approval ID: 2010022). Male db/db were obtained from Jackson Laboratories (Bar Harbor, ME, USA). We used db/db mice at the age of 12-16 weeks (50-60 g) when they had developed overt diabetes.
Db/db mice were randomly allocated into the following groups with n = 30 each: (i) Db/db + sham group (Sham); (ii) Db/db + I/R group (I/R); (iii) Db/db + OSM + I/R group (OSM); (iv) Db/db + TNP + I/R (TNP) and (v) Db/db + TNP + OSM + I/R (TNP + OSM). Before constructing cardiac I/R model, OSM (60 ng/g; ≥97%, Sigma Aldrich, St. louis, MO, USA) was injected intraperitoneally for 14 days. TNP (IP6K inhibitor, 10 mg/kg; ≥95%, Sigma-Aldrich) was injected via the tail vein for 14 days. TNP + OSM group received TNP injection 10 min. before OSM injection for 14 days. All the reagents were dissolved in DMSO. The sham group and the I/R group received the same volume of DMSO for 14 days. The time interval between the last injection and ischaemiareperfusion was 1 hr. Blood glucose concentration was determined by using a reflectance metre (Accu-Chek, Roche Diagnostics GmbH, Mannheim, Germany). Food intake and bw were recorded regularly.
129-Osmr tm1.1Nat /J mice were purchased from Jackson Laboratories which possess loxP sites on either side of the second exon (first coding exon) in the OSM receptor (Ob) gene. 129-Osmr tm1.1Nat /J mice were crossed with C-Tg(CMV-cre)1Cgn/J mice (Jackson Laboratories) to knockout OSM receptor Ob. Real-time PCR (RT-PCR) was used to screen Ob À/À mice and Ob +/+ mice.
Diabetes was induced in male Ob À/À and Ob +/+ mice by intraperitoneal injections (i.p.) of STZ (50 mg/kg, STZ was dissolved in 0.1 M citrate buffer, pH 4.5) as previously described [19]. All the mice were fed with high glucose and high-fat diet after STZ injection. Blood glucose concentration was determined every week after STZ injection. Random blood glucose was tested in each mouse for three times. All of these three values ≥16.7 mmol/l were considered as a cut-off point for diabetes. Two months after STZ administration, diabetic Ob À/À mice and diabetic Ob +/+ mice were subjected to myocardial I/R injury.

Construction of I/R injury animal model and hemodynamic evaluation
I/R injury animal model was constructed by LAD ligation for 30 min. followed by 3 hrs reperfusion as previously described [20]. Cardiac function was determined by invasive hemodynamic evaluation methods as previously described [20].

Measurement of myocardial infarct size
Myocardial infarct size was evaluated by Evans Blue/TTC staining as previously described [20].

Determination of myocardial apoptosis
Myocardial apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) staining and caspase-3 activity assay as previously described [20].

Determination of cardiac function
Echocardiography was conducted at 24 hrs after I/R injury as previously described [20].

Mitochondrial calcium retention capacity
The mitochondrial calcium retention capacity (mCRC) was determined as the capacity of mitochondria to uptake calcium before permeability transition, to test the sensitivity of the mitochondrial permeability transition pore (mPTP) opening to calcium.

ROS production and Manganese superoxide dismutase activity
The production of Reactive oxygen species (ROS) was measured in frozen tissue by electron paramagnetic resonance (EPR) spectroscopy according to Mellin's methods [21]. The ROS levels were expressed in arbitrary units per milligram of wet tissue. Manganese superoxide dismutase (MnSOD) was assayed as Vives-Bauza has previously described [22] and expressed in unit/mg.
In vitro citrate synthase, chain complex activities and ATP content Citrate synthase (CS) and electron transport chain complex activities (Complex I, II, III, IV and IV) were measured using a commercially available CS activity assay kit (Sigma-Aldrich). The ATP content of the myocardium was measured using an ATP bioluminescent assay kit (Sigma-Aldrich) according to the standard protocols.

Statistical analysis
Continuous variables that approximated the normal distribution were expressed as means AE SD. Comparison between groups were subjected to ANOVA followed by Bonferroni correction for post hoc t-test. Data expressed as proportions were assessed with a chi-squared test. Two sided tests have been used throughout, and P < 0.05 were considered statistically significant. SPSS software package version 14.0 (SPSS, Chicago, IL, USA) was used for data analysis.

OSM administration alleviates cardiac I/R injury in db/db mice by inhibition of IP7 production
Lactate dehydrogenase (LDH) and creatine kinase-myocardium isoenzyme (CK-MB), biochemical markers of myocyte injury, were significantly decreased in the OSM treated group as compared with the I/R group ( Fig. 1A and B). Representative images of infarct size as stained by Evans Blue and TTC were shown in Figure 1C. OSM administration significantly decreased infarct size at 3 hrs after I/R injury compared with the I/R group (Fig. 1D). The ratio of area at risk (AAR) to left ventricle (LV) area had no statistical difference between groups, indicating that LAD ligature was reproducibly performed at the same level (Fig. 1E). OSM treatment resulted in a noticeable decrease in MPO activity, IL-1a and TNF-a release and IP7 production compared with the I/R group ( Fig. 1F-I). Echocardiography evaluated at 24 hrs after I/R injury revealed that OSM significantly enhanced LVEF, decreased LVESV as compared with the I/R group ( Fig. 1J-M). Hemodynamic measurements performed at 3 hrs after I/R injury indicated that the AELV dp/dt max were increased in the OSM group as compared with the I/R group ( Fig. 1N and O).
To determine the causative role of OSM/IP7 signalling in cardiac I/R injury, we treated the db/db mice systemically with TNP followed by OSM before cardiac I/R injury. Interestingly, OSM did not exhibit additional protective effects against cardiac I/R injury as compared with the TNP treated group.

Ob (OSM receptor) knockout exacerbates myocardial I/R injury, increases IP7 production in diabetic mice
To elucidate the impact of OSM on cardiac I/R injury in the genetically OSM receptor Ob free situation in diabetic mice, the Ob À/À and its littermate control mice (Ob +/+ ) were injected by STZ to construct diabetes model and were subjected to cardiac I/R injury. Less I/R injury was manifested by lower levels of serum LDH and CK-MB in Ob +/+ mice ( Fig. 2A and B). With similar AAR/LV, the infarct size was significantly smaller in Ob +/+ mice as compared with Ob À/À mice ( Fig. 2C-E). The diabetic Ob +/+ mice exhibited reduced MPO activity (Fig. 2F), decreased release of IL-1a and TNF-a ( Fig. 2G and H) and decreased production of IP7 (Fig. 2I) after I/R injury. In parallel, we observed improved cardiac function, as indicated by elevated LVEF, decreased LVESV and LVEDV in Ob +/+ mice ( Fig. 2J-M). This protection was also demonstrated by increased AELV dp/dt max ( Fig. 2N and O) in Ob +/+ mice as compared with Ob À/À mice. These results suggest that OSM plays an important role in reducing myocardial I/R injury by inhibition of IP7 production.
OSM inhibits cardiomyocyte apoptosis induced by cardiac I/R injury in db/db mice by reducing IP7 production TUNEL-positive cardiomyocytes were less frequently observed in the OSM group as compared with the I/R group. Similarly, TNP treatment  remarkably decreased the number of TUNEL-positive cardiomyocytes compared with the I/R group (Fig. 3A and B). Concomitantly, caspase-3 activity determined by a caspase colorimetric assay, caspase-3 and cleaved caspase-3 expression evaluated by Western blot was down-regulated by OSM, TNP or TNP+OSM treatment (Fig. 3C-E). After 3 hrs of reperfusion, western blot analysis revealed that OSM or TNP treatment increased the phosphorylation state of Akt (T308, S473), TSC2, S6K1, S6 and BAD while decreased the phosphorylation state of GSK3b. Furthermore, OSM or TNP administration decreased Bax/Bcl-2 ratio in cardiac tissue that were exposed to I/R injury ( Fig. 3F-J).
Ob knockout enhances cardiomyocyte apoptosis in diabetic mice who underwent cardiac I/R injury TUNEL-positive cardiomyocytes, caspase-3 and cleaved caspase-3 expression were remarkably reduced in diabetic Ob +/+ mice who underwent cardiac I/R injury as compared with the Ob À/À group (Fig. 4A-E). Diabetic Ob +/+ mice who underwent cardiac I/R injury exhibited much higher the phosphorylation state of Akt(T308, S473), TSC2, S6K1, S6, BAD and decreased the phosphorylation state of GSK3b. The pro-to anti-apoptotic protein (Bax/Bcl-2) ratio was significantly decreased in the Ob +/+ group (Fig. 4F-I).
OSM improves mitochondrial biogenesis and mitochondrial function in db/db mice who underwent cardiac I/R injury by reducing IP7 production Transmission electron microscopy revealed that OSM or TNP treatment alleviated disorganized mitochondria architecture in db/db mice which underwent cardiac I/R injury (Fig. 5A). Mitochondrial DNA content (Fig. 5B), ATP content (Fig. 5C), CS activity (Fig. 5D), and complex I/II/III/IV/V activities (Fig. 5E) in the ischaemic myocardium were significantly enhanced in the OSM or TNP treated group as compared to the I/R group. OSM or TNP administration increased mCRC (Fig. 5F), indicating that sensitivity to calciuminduced mPTP opening was decreased. ROS levels (Fig. 5G) assessed by EPR spectroscopy and mitochondrial MnSOD activity (Fig. 5H) were decreased in the OSM and TNP treated group as compared to the I/R group.
Western blot analysis revealed that OSM or TNP administration increased the phosphorylation state of adenine mononucleotide protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), the expression of Nrf-1 and Tfam. The acetylation state of PGC-1a was decreased in the OSM or TNP treated group (Fig. 5I-K).

Ob knockout impairs mitochondrial biogenesis and function in diabetic mice who underwent cardiac I/R injury
Ob knockout mice exhibited increased mitochondria morphological defects (Fig. S1A). The Ob À/À group showed decreased Mitochondrial DNA content (Fig. S1B), ATP content (Fig. S1C), CS activity (Fig. S1D) and complex I/II/III/IV/V activities (Fig. S1E) as compared to the Ob +/+ group. The mCRC was decreased (Fig. S1F), ROS levels (Fig. S1G) and mitochondrial MnSOD activity (Fig. S1H) were increased in the Ob À/À group as compared to the Ob +/+ group. Together, these results suggested that Ob knockout impaired mitochondrial biogenesis and mitochondrial V dysfunction in diabetic mice which underwent cardiac I/R injury. Western blot analysis showed that Ob knockout decreased the phosphorylation state of AMPK and ACC, the expression of Nrf-1 and Tfam. The acetylation state of PGC-1a was increased in the Ob À/À group ( Fig. S1I and J).

Ob knockout impairs glucose homoeostasis and insulin sensitivity in diabetic mice which underwent cardiac I/R injury
Ob knockout led to a marked impairment in GTT. Glucose concentrations during the intraperitoneal GTT were significantly higher in Ob À/À mice as compared to the Ob +/+ mice (Fig. S2A). The insulin sensitivity evaluated by intraperitoneal ITT manifested that Ob knockout significantly decreased the blood glucose-lowering effect of injected insulin (Fig. S2B). The Ob +/+ mice required a higher rate of exogenous glucose infusion to maintain euglycaemia, indicating enhanced whole body sensitivity to the insulin infusion (Fig. S2C). Consistently, Ob À/À mice exhibited significantly decreased glucose disposal rate (Fig. S2D). Ob knockout decreased insulin-stimulated glucose transport activity in the soleus, gastrocnemius and epididymal white adipose tissue (Fig. S2E). To obtain corroborative evidence of insulin sensitivity in muscle, we investigated insulin signalling by analysing  the tyrosine phosphorylation state of IRS1, the phosphorylation state of Akt (p-T308, p-S473) and GSK3b (Fig. S2F). Western blot analysis revealed decreased phosphorylation state of IRS1 and Akt, increased phosphorylation state of GSK3b in the muscles of Ob À/À mice (Fig. S2G), further supporting the capacity of Ob knockout to downregulate insulin signalling in skeletal muscle.

Discussion
This study provides the first direct evidence for a role of OSM in regulating apoptosis, mitochondrial biogenesis and insulin sensitivity in diabetic mice which underwent myocardial I/R injury. OSM receptor Ob knockout exacerbated cardiac I/R injury, increased IP7 production, enhanced cardiomyocyte apoptosis, impaired mitochondrial biogenesis, glucose homoeostasis and insulin sensitivity in diabetic mice which underwent cardiac I/R injury. Interestingly, inhibition of IP7 production by TNP exhibited similar effects as OSM, the combination of OSM and TNP was comparable to each agent alone, indicating a causative role of OSM/IP7 signalling in cardiac I/R injury in diabetic mice. The mechanism of OSM on cardiac I/R injury in diabetic mice is partly associated with IP7/Akt and AMPK/PGC-1a pathway.
Blocking the cardiomyocyte apoptosis process could prevent the loss of contractile cells, minimize cardiac injury induced by I/R injury and therefore slow down the occurrence of heart failure [23]. Our study demonstrated that OSM administration significantly reduced cardiomyocyte apoptosis induced by cardiac I/R injury in db/db mice. The diabetic Ob +/+ mice exhibited decreased cardiomyocyte apoptosis as compared to the diabetic Ob À/À mice who underwent I/R injury. Furthermore, OSM pre-treatment inhibited IP7 production and TNP administration exhibited similar anti-apoptotic effects as OSM, suggesting that IP7 may serve as an effective downstream molecule of OSM.
It has been reported that PI3K/Akt pathway activation leads to BAD phosphorylation and may thereby suppress cell apoptosis [20]. In the present study, the anti-apoptotic effects of OSM and TNP were also associated with activation of PI3K/Akt/BAD pathway. The Bcl-2 family is a key regulator of physiological and pathological apoptosis. The relative ratio of proapoptotic proteins (i.e., Bax) to antiapoptotic proteins (i.e., Bcl-2) plays a key role in determining cell survival or death. It has been demonstrated that the high ratio of Bax/Bcl-2 is associated with greater vulnerability to apoptotic activation [24]. In the present study, OSM or TNP treatment decreased Bax/Bcl-2 ratio. On the contrary, the Diabetic Ob À/À mice exhibited much higher Bax/ Bcl-2 ratio, indicating that OSM may regulate cardiomyocyte apoptosis by IP7/Akt/BAD pathway.
Potential roles of mitochondrial dysfunction in diabetes and cardiac I/R injury have recently emerged from in vitro studies. Modulating mitochondrial survival pathways will also affect apoptosis and necrosis [25]. In accordance, our study identified significant morphological, biogenesis and function defects in mitochondria after cardiac I/R injury in db/db mice. OSM or TNP administration increased, while OSM receptor Ob knockout decreased Mitochondrial DNA content, CS activity, complex I/II/III/IV/V activities and ATP production. OSM did not exhibit additional protective effects against mitochondria morphology, biogenesis abnormality and mitochondrial dysfunction when administered after TNP treatment. This indicates that IP7 may serve as a mediator of the effects of OSM.
Mitochondria are the main source of ROS, which are fundamental factors in the development of diabetic complications [9,26,27]. Therefore, eradicating mitochondrial ROS has become important to ameliorate complications related with diabetes [25]. We found lower levels of ROS production in the OSM or TNP treated group, while Ob knockout increased ROS production. In addition, the OSM group exhibited a down-regulation of mitochondrial MnSOD activity and a decreased sensitivity of mPTP opening as demonstrated by increased mCRC. Both can be regarded as indirect evidence for decreased mitochondria-targeted ROS. On the contrary, Ob knockout increased MnSOD activity and sensitivity of mPTP opening.
Adenine mononucleotide protein kinase is a major regulator of mitochondrial biogenesis, which promotes mitochondrial biogenesis through PGC1-a [28]. Expression of PGC-1 is reported to be undermined in diabetic and insulin-resistant human subjects, respectively [29]. ACC is the downstream signalling substrate of AMPK. Consistent with those interpretations, OSM or TNP administration increased the phosphorylation state of AMPK and ACC, the expression of Nrf-1 and Tfam. The acetylation state of PGC-1a was decreased in the OSM, TNP or TNP+OSM group. The results suggested that OSM may regulate mitochondrial biogenesis by AMPK/ PGC-1a pathway.
Insulin resistance is associated with mitochondrial dysfunction [30]. Mitochondrial dysfunction, including mitochondrial biogenesis abnormality and excessive oxidative stress, is related to the pathological effects of insulin resistance in different tissues. Modulating mitochondrial function may improve insulin resistance and reduce subsequent cardiac mortality. Insulin signalling activation leads to PI3K activation and several downstream serine kinases, including protein kinase B (Akt), protein kinase C, etc., all of which eventually modulate the biological and pleiotropic metabolic actions of insulin [31]. In the present study, Ob knockout led to a marked impairment in glucose tolerance and whole body insulin sensitivity in diabetic mice which underwent cardiac I/R injury. Euglycemic-hyperinsulinemic clamp experiments demonstrated decreased glucose disposal in peripheral tissues in Ob knockout mice. To investigate the underlying mechanism of Ob knockout in impairment of insulin sensitivity in muscle, insulin signalling was analysed. Western blot analysis revealed decreased tyrosine phosphorylation state of IRS1, decreased the phosphorylation state of Akt(T308, S473) in the muscles of Ob À/À mice, further supporting the capacity of Ob knockout to down-regulate insulin signalling. The effects of OSM and TNP in protecting against cardiac I/R injury in diabetic mice were investigated in db/db mice. For the Ob À/À and Ob +/+ mice, we injected STZ and fed the mice with high glucose and high fat diet to induce diabetic model. Interestingly, the results were consistent between those two animal models.   The pro-to anti-apoptotic protein (Bax/ Bcl-2) ratio was increased in the Ob À/À group. *P < 0.01 versus Sham, # P < 0.01 versus I/R, § P < 0.01 versus OSM.

Conclusions
This study provides the first direct evidence for a role of OSM in regulating cardiomyocyte apoptosis, mitochondrial biogenesis and insulin sensitivity in diabetic mice undergoing myocardial I/R injury. The effects of OSM are partly associated with IP7/Akt and AMPK/PGC-1a pathway. Moreover, our results suggest that OSM is a potential novel therapeutic target for preventing myocardial I/R injury in diabetic mice.