Diabetes Volume 64, October 2015 3573 Genaro A. Ramirez-Correa,1 Junfeng Ma,2 Chad Slawson,3 Quira Zeidan,2 Nahyr S. Lugo-Fagundo,1 Mingguo Xu,1 Xiaoxu Shen,4 Wei Dong Gao,4 Viviane Caceres,5 Khalid Chakir,5 Lauren DeVine,2 Robert N. Cole,2 Luigi Marchionni,6 Nazareno Paolocci,5 Gerald W. Hart,2 and Anne M. Murphy1 Removal of Abnormal Myofilament O-GlcNAcylation Restores Ca2+ Sensitivity in Diabetic Cardiac Muscle Diabetes 2015;64:3573–3587 | DOI: 10.2337/db14-1107 Contractile dysfunction and increased deposition of In diabetic cardiomyopathy, the contractile and electro- O-linked b-N-acetyl-D-glucosamine (O-GlcNAc) in car- physiological properties of the cardiac muscle are altered diac proteins are a hallmark of the diabetic heart. How- (1). Prior studies have mainly focused on alterations in ever, whether and how this posttranslational alteration Ca2+ handling (2–4). However, these perturbations alone contributes to lower cardiac function remains unclear. unlikely account for lower force production and altered COMPLICATIONS fi b Using a re ned -elimination/Michael addition with tan- relaxation typically found in the heart of patients with dem mass tags (TMT)–labeling proteomic technique, we diabetes (5,6). Indeed, the intrinsic properties of cardiac show that CpOGA, a bacterial analog of O-GlcNAcase myofilaments appear to be altered too (7,8). More specif- (OGA) that cleaves O-GlcNAc in vivo, removes site- 2+ 2+ fi specific O-GlcNAcylation from myofilaments, restoring ically, Ca sensitivity (ECa 50), a measure of myo la- 2+ Ca2+ sensitivity in streptozotocin (STZ) diabetic cardiac ment force production at near physiological Ca levels, – muscles. We report that in control rat hearts, O-GlcNAc is reduced in the heart of patients with diabetes (9 11). 2+ and O-GlcNAc transferase (OGT) are mainly localized at Yet the mechanisms responsible for Ca desensitization the Z-line, whereas OGA is at the A-band. Conversely, in in diabetic hearts remain incompletely understood. diabetic hearts O-GlcNAc levels are increased and OGT O-GlcNAcylation is a posttranslational modification (PTM) and OGA delocalized. Consistent changes were found in linked to glucose metabolism and centrally involved in reg- human diabetic hearts. STZ diabetic hearts display in- ulating cellular homeostasis (12). This PTM consists of creased physical interactions of OGA with a-actin, the addition of single O-linked b-N-acetyl-D-glucosamine tropomyosin, and myosin light chain 1, along with re- (O-GlcNAc) sugar to serine (Ser) and threonine (Thr) res- duced OGT and increased OGA activities. Our study is idues of nuclear and cytoplasmic proteins. The reaction the first to reveal that specific removal of O-GlcNAcylation is catalyzed by O-GlcNAc transferase (OGT), whereas restores myofilament response to Ca2+ in diabetic hearts O-GlcNAc removal is under the control of O-GlcNAcase and that altered O-GlcNAcylation is due to the subcellular (OGA) (12). Excessive O-GlcNAcylation results from redistribution of OGT and OGA rather than to changes in glucose- or other nutrient-induced overload of the hexos- their overall activities. Thus, preventing sarcomeric OGT amine biosynthesis pathway (HBP). Alterations in HBP and OGA displacement represents a new possible strat- are increasingly recognized as a major contributing factor egy for treating diabetic cardiomyopathy. for insulin resistance (12) and “glucose toxicity” during 1Division of Cardiology, Department of Pediatrics, Johns Hopkins University 6Department of Oncology, Johns Hopkins University School of Medicine, Balti- School of Medicine, Baltimore, MD more, MD 2 Department of Biological Chemistry, Johns Hopkins University School of Medi- Corresponding author: Genaro A. Ramirez-Correa, [email protected]. cine, Baltimore, MD Received 1 August 2014 and accepted 14 May 2015. 3Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS G.A.R.-C. and J.M. contributed equally to this work. 4Department of Anesthesiology and Critical Care Medicine, Johns Hopkins Uni- © 2015 by the American Diabetes Association. Readers may use this article as versity School of Medicine, Baltimore, MD long as the work is properly cited, the use is educational and not for profit, and 5Division of Cardiology, Department of Medicine, Johns Hopkins University School the work is not altered. of Medicine, Baltimore, MD See accompanying article, p. 3339. 2+ 3574 OGA Restores Cardiac Muscle ECa 50 in Diabetes Diabetes Volume 64, October 2015 diabetes. Similar to phosphorylation, O-GlcNAcylation is Immunofluorescence Confocal Microscopy a widely distributed and highly dynamic PTM (12). How- Cold acetone–fixed cryosections (7–8 mm) from rat or ever, unlike phosphorylation, which is regulated by a myriad human (BioChain Inc., Office of Human Research Protec- of kinases and phosphatases, the extent of O-GlcNAcylation tion registered IRB00008283) myocardium were blocked relies on two enzymes only, specifically OGT and OGA. OGT and incubated overnight with a primary antibody against substrate specificity is regulated by transient protein-to- O-GlcNAc (CTD110.6). In addition, isolated skinned myo- protein interactions that take place primarily at its tetratri- cytes were obtained from flash-frozen myocardium by copeptide repeat (TPR) domain (12) (Fig. 7A). Often homogenization in 0.03% Triton X-100 at low speed as OGT and OGA interact with each other and/or are found previously described (28), seeded on eight chamber slides forming a holoenzyme complex with protein phospha- coated with 40 mg/mL laminin (Invitrogen), and fixed in tases and kinases (12). Modifications of Ser and Thr by 4% formaldehyde-methanol–free ultrapure (Polysciences O-GlcNAc occur in myofilaments, and the addition of ex- Inc.). Cryosections or isolated skinned myocytes were ogenous N-acetyl-D-glucosamine (GlcNAc) alters myofila- blocked and incubated overnight with OGA O-GlcNAcase ment response to Ca2+ (13,14). In addition, manipulation (345), OGT (AL-25) (29), and anti–a-actinin (Sigma-Aldrich) of cardiac O-GlcNAc levels influences Ca2+ cycling kinetics at 1 mg/mL. Secondary antibodies were Alexa 647 goat anti- (4) and mitochondrial rates of respiration in diabetes mouse IgM (m-chain) for O-GlcNAc, Alexa 647 goat anti- (2,15) and functional recovery after ischemia-reperfusion rabbit IgG for OGT, Alexa 594 goat anti-chicken IgY for injury (16,17) or chronic pressure-overload (18–21). OGA, and Alexa 488 goat anti-mouse IgG for a-actinin. Despite all of this evidence, whether and how altered Prolong antifade with DAPI (Invitrogen) was used for O-GlcNAcylation contributes to myofilament dysfunc- mounting. Images were acquired on a Zeiss 710 LSM up- tion in diabetic cardiomyopathy (7,8,22–24) is currently right microscope using a 325 or a 363 water-immersion unclear. objective (Nikon) and analyzed with Zen 9 Leica Zeiss soft- Here we proved that specific removal of O-GlcNAc ex- ware tools. cess from diabetic myofilaments ameliorates contractile Double Immunoelectron Microscopy dysfunction by linking improvement in force-Ca2+ rela- For gold immunolabeling, goat anti-rabbit or goat anti- tionships to site-specific O-GlcNAc changes. We also mouse were labeled with 12-nm-diameter particles to determined a potential mechanism leading to altered detect anti-OGT (AL-34) or anti–O-GlcNAc (CTD 110.6), O-GlcNAcylation by comparing the status and sarcomeric and goat anti-chicken was labeled with 6-nm-diameter par- distribution patterns of OGT and OGA in the heart of rats ticles to detect anti-OGA (345). Labeled ultrathin sections with streptozotocin (STZ)-induced type 1 diabetes with (60- to 90-nm thick) were examined under the transmis- that found in controls. sion electron microscope (Hitachi 7600 TEM, Tokyo, Japan), and 8–11 random field pictures were used for RESEARCH DESIGN AND METHODS quantification of OGT, OGA, and O-GlcNAc immunolabeled All animal protocols in this study were performed in gold particles with ImageJ software (National Institutes of accordance with institutional guidelines and approval of Health [NIH]). the Johns Hopkins University School of Medicine Insti- tutional Animal Care and Use Committee. Coimmunoprecipitation for OGT and OGA For immunoprecipitation studies, anti-OGT (AL-28) or Type 1 Diabetes Rat Model anti-OGA (345) (1 mg total) antibodies were added to Type 1 diabetes was induced in male Sprague-Dawley rats 0.5 mg/mL protein samples. Immunoprecipitates were (Charles River) by an intraperitoneal injection of STZ then separated by SDS-PAGE, transferred to polyvinyli- (65 mg/kg). Control animals were injected with vehicle only. dene fluoride membranes (Millipore, Bedford, MA) for Animals were killed 6–8 weeks after induction of diabetes. antibody probing against a-cardiac actin (Sigma-Aldrich), At the moment of tissue harvest, STZ diabetic animals had a-tropomyosin (Tm; Sigma-Aldrich), and anti-myosin blood glucose of 693.5 6 61.5 mg/dL and controls had light chain (MLC) 1 (Clone 1LC-14, Spectral Diagnos- 123.9 6 9.5 mg/dL. tics). Between different antibodies blots were stripped Isolated Skinned Fiber Studies for 1 h at 25°C in 200 mmol/L glycine (pH 2.5; Sigma- For skinned cardiac muscles studies, muscles were isolated Aldrich) (29). and mounted as previously described (25,26). Varied Ca2+ 2+ OGT and OGA Activity Assays concentrations [Ca ]o were achieved by mixing the relaxing solutionandactivatingsolutioninvariousratios.After OGT Assays 2+ reaching the highest [Ca ]o concentration, trabeculae were Heart homogenates were separated in cytosolic or myo- washed in relaxing solution and incubated at room temper- filament fractions, desalted, and subjected to OGT activity ature 1 h in 1 mg/mL CpOGA (27) diluted in relaxing solu- assays as described (30). Activity counts in disintegration tion.Afterward,anewCa2+ activation protocol was repeated. per minute (dpm) were normalized to total protein con- 2+ Steady-state force-[Ca ]o relationships were determined ex- tent (dpm/mg), and then background activity without CKII perimentally and fittoamodified Hill equation (25,26). peptide was subtracted (Fig.