Hyperglycemia Abrogates Ischemic Postconditioning Cardioprotection by Impairing Adipor1/Caveolin-3/STAT3 Signaling in Diabetic Rats
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942 Diabetes Volume 65, April 2016 Haobo Li,1,2 Weifeng Yao,2 Zipeng Liu,2,3 Aimin Xu,1,4 Yu Huang,1,5 Xin-liang Ma,6 Michael G. Irwin,2 and Zhengyuan Xia1,2 Hyperglycemia Abrogates Ischemic Postconditioning Cardioprotection by Impairing AdipoR1/Caveolin-3/STAT3 Signaling in Diabetic Rats Diabetes 2016;65:942–955 | DOI: 10.2337/db15-0782 Signal transducer and activator of transcription 3 Myocardial infarction is a major perioperative complica- (STAT3) activation is key for ischemic postconditioning tion in patients with diabetes (1). Reperfusion therapies (IPo) to attenuate myocardial ischemia-reperfusion restore coronary flow but may cause lethal tissue injury, injury (MIRI), but IPo loses cardioprotection in diabe- called “reperfusion injury.” Ischemic postconditioning tes in which cardiac STAT3 activation is impaired and (IPo), the phenomenon that brief repetitive episodes of adiponectin (APN) reduced. We found that IPo increased ischemia and reperfusion (IR) at the immediate onset of postischemic cardiomyocyte-derived APN, activated reperfusion, can protect the hearts against myocardial mitochondrial STAT3 (mitoSTAT3), improved mitochon- IR injury (MIRI) (2), which needs to activate signal drial function, and attenuated MIRI in wild-type but not 2/2 transducer and activator of transcription 3 (STAT3) (3). in APN knockout (Adipo ) mice subjected to 30 min However, how IPo activates STAT3 remains unclear. In coronary occlusion, followed by 2 or 24 h of reperfusion. addition,heartsfromsubjectswithdiabetesarelessor Hypoxic postconditioning–induced protection against not sensitive to IPo, and the mechanism is unknown (4). hypoxia/reoxygenation injury was lost in Adipo2/2 car- The reduction of myocardial STAT3 activation in diabetes diomyocytes but restored by recombinant APN, but SIGNAL TRANSDUCTION and the subsequent reduction of myocardial nitric oxide this APN beneficial effect was abolished by specific (NO) bioavailability (5) may be primarily responsible for STAT3 or APN receptor 1 (AdipoR1) gene knockdown, or caveolin-3 (Cav3) disruption. APN activated cardiac the loss of myocardial sensitivity to IPo (4). STAT3 and restored IPo cardioprotection in 4-week di- Adiponectin (APN), an adipocyte-derived plasma pro- abetic rats where AdipoR1 and Cav3 were functionally tein with antidiabetic properties, protects against MIRI interactive but not in 8-week diabetic rats whose car- by reducing myocardial oxidative/nitrative stress and diac Cav3 was severely reduced and AdipoR1/Cav3 activating endothelial nitric oxide synthase (eNOS) and signaling impaired. We concluded that IPo activates increasing NO bioavailability (6), which requires the in- mitoSTAT3 through APN/AdipoR1/Cav3 pathway to teraction of caveolin-3 (Cav3), the structural protein for confer cardioprotection, whereas in diabetes, IPo loses caveolae, which are flask-shaped plasma membrane invag- cardioprotection due to impaired APN/AdipoR1/Cav3 inations, and APN receptor 1 (AdipoR1), the predominant signaling. Therefore, effective means that may concom- APN receptor expressed in cardiomyocytes (7). In cultured itantly activate APN and repair APN signaling (i.e., adult mouse cardiac fibroblasts, APN activates STAT3 (8). AdipoR1/Cav3) in diabetes may represent promising However, whether APN can activate or facilitate the acti- avenues in the treatment of MIRI in diabetes. vation of STAT3 in cardiomyocytes in the context of MIRI 1State Key Laboratory of Pharmaceutical Biotechnology, The University of Hong Received 9 June 2015 and accepted 12 December 2015. Kong, Hong Kong, China This article contains Supplementary Data online at http://diabetes 2 Department of Anesthesiology, The University of Hong Kong, Hong Kong, China .diabetesjournals.org/lookup/suppl/doi:10.2337/db15-0782/-/DC1. 3Centre for Genomic Sciences, The University of Hong Kong, Hong Kong, China © 2016 by the American Diabetes Association. Readers may use this article as 4Department of Medicine, The University of Hong Kong, Hong Kong, China long as the work is properly cited, the use is educational and not for profit, and 5Institute of Vascular Medicine and Li Ka Shing Institute of Health Sciences, The the work is not altered. Chinese University of Hong Kong, Hong Kong, China 6Department of Emergency Medicine, Thomas Jefferson University, Philadelphia, PA See accompanying article, p. 826. Corresponding author: Zhengyuan Xia, [email protected]. diabetes.diabetesjournals.org Li and Associates 943 in nondiabetic and especially in diabetic conditions, a Evans blue/2,3,5-triphenyl-2H-tetrazolium chloride (TTC) pathological condition that is accompanied with increased staining, as previously described (9), and the operators oxidative stress, is unknown. Nevertheless, the properties were blinded to the information of study design and in- of APN (i.e., activating STAT3 and enhancing NO bioavail- tervention. Global cardiac functions were monitored by ability) suggest that APN may play significant roles in the using a pressure-volume conductance catheter and ana- cardioprotective actions of IPo. Furthermore, the finding lyzed using LabChart 8 software (ADInstruments, Colorado that antioxidant treatment enhanced STAT3 activation in Springs, CO), as we described previously (19). the heart of diabetic rats through APN signaling (9) sug- gests that malfunction of APN might be a key mechanism Detection of Myocardial Apoptosis that rendered the diabetic heart less or not responsive to Myocardial apoptosis was determined by TUNEL staining IPo cardioprotection. In addition, cardioprotective effects using an In Situ Cell Death Detection Kit (Roche Diagnostics of APN were diminished in high-fat diet–induced diabetes GmbH, Mannheim, Germany), as previously described (10,11), in which Cav3 is downregulated (12). This, to- (20). The sections were observed in the light microscope fi by an investigator who was initially blinded to treatment gether with our recent nding that Cav3 is disrupted (13) fi and STAT3 inactivated (9), which deranged eNOS signal- groups. Five randomly selected elds of each slide were ing in diabetic myocardium with concomitantly reduced analyzed, and the apoptotic index was calculated as a APN (9), indicates that APN signaling (e.g., AdipoR1/Cav3) percentage of apoptotic nuclei to total nuclei. is impaired in diabetes. Although AdipoR1 (14,15) and Electron Microscopic Analysis STAT3 (16) have both been shown to be important procell Heart tissues were fixed with 2.5% glutaraldehyde in 0.1 survival factors in multiple cells, their potential interplay mol/L phosphate buffer (pH 7.4), followed by 1% OsO4. in affecting postischemic cell survival in general and spe- After dehydration, thin sections were stained with uranyl fi ci cally in affecting the myocardial responsiveness to IPo acetate and lead citrate for observation under a JEM cardioprotection in diabetes is unknown. We hypothe- 1011CX electron microscope. Images were acquired digitally. sized that the impairment of cardiac APN signaling is re- sponsible for the inactivation of STAT3 in diabetes that Induction of Diabetes and APN Adenovirus Infection disables the ability of the diabetic heart to respond to IPo Type 1 diabetes was induced via signal tail-vein injection and that an intact or adequate AdipoR1/Cav3 interaction of streptozocin (STZ) (65 mg/kg; Sigma-Aldrich, St. Louis, is critical for IPo to activate STAT3 via APN. MO), as previously described (21). One week after STZ injection, rats exhibiting hyperglycemia (blood glucose $ RESEARCH DESIGN AND METHODS 16.7 mmol/L) were considered diabetic and subjected to outlined experiments. Recombinant APN adenovirus Experimental Animals (1*109 plaque-forming units) was used to overexpress APN Male Sprague-Dawley rats (250 6 8g,6–8 weeks) were in vivo, and luciferase was used as the control, which were obtained from the Laboratory Animal Unit (The Univer- 2 2 injected by tail vein into rats 1 week before inducing IR, sity of Hong Kong). Male APN knockout (Adipo / ) mice as previously described (19). (6–8 weeks) with a C57BL/6J background and age- matched wild-type (WT) control mice with the same ge- Measurement of Mitochondrial Respiratory Chain netic background were generated as previously described Complex Activities (17). All of the experiments were conducted in adherence At the end of experiments, heart tissues were immediately with the National Institutes of Health Guide for the Care collected for cardiac mitochondria isolation according and Use of Laboratory Animals and were approved by the to the manufacturer’s protocol per the mitochondria ex- Institutional Animal Care and Use Committee. traction kit (Thermo Fisher Scientific, Chicago, IL). The resulting mitochondrial pellets were resuspended in Tris- MIRI and IPo In Vivo hydroxymethyl aminomethane solution (pH 8.0) and kept After anesthesia, animals were randomized to receive at 280°C for subsequent measurements of mitochondrial sham operation, myocardial IR, or IPo. Myocardial IR was respiratory chain complex activities (complex I-V), as induced by temporarily exteriorizing the heart via a left previously described (22). thoracic incision, and the left anterior descending coronary artery was occluded for 30 min, followed by Determination of 15-F2t-Isoprostane, NO, reperfusion for 2 h (to assess postischemic mRNA and Nitrotyrosine, ATP Content, and Reactive Oxygen protein expressions of cardiac APN and other signaling Species molecules) or 24 h (to assess postischemic cardiac func- Cardiac tissue (area at risk) was rinsed and homogenized. tion, myocardial injury,