Differential Protein Acetylation Mediates Mitochondrial Localization and Pentameric Aggregation of SOD2 in the Murine Heart of SOD2-Tg

Differential Protein Acetylation Mediates Mitochondrial Localization and Pentameric Aggregation of SOD2 in the Murine Heart of SOD2-Tg

319 Nitric Oxide-Mediated Angiogenesis Is Improved by β3 Adrenergic Receptor Stimulation Kristen J Bubb1, Owen Tang1, Zoe Clayton2, Thomas Hansen1, Sa...

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319 Nitric Oxide-Mediated Angiogenesis Is Improved by β3 Adrenergic Receptor Stimulation Kristen J Bubb1, Owen Tang1, Zoe Clayton2, Thomas Hansen1, Sanjay Patel2, and Gemma A Figtre,1,3 1 Kolling Institute, Sydney, Australia, 2Heart Research Institute and University of Sydney, Australia, 3Cardiology, Royal North Shore Hospital, Sydney, Australia Background: Angiogenesis is essential for vascular regeneration following ischemic disease and nitric oxide (NO) is a key regulator of this. β3 adrenergic receptor (β3AR) agonists are known to activate endothelial nitric oxide synthase (eNOS) and we have recently demonstrated that this is at least partially mediated by their ability to decrease glutathionylation-mediated uncoupling of the enzyme, restoring NO production, and decreasing superoxide generation. We hypothesized that β3AR stimulation would promote angiogenesis. Methods and Results: Hindlimb ischemia in mice caused a drastic reduction in limb perfusion, with a gradual reperfusion indicative of angiogenesis. Mice treated with a β3AR agonist, CL 316, 243 (1mg/kg/day subcutaneously via osmotic minipump) showed a more rapid improvement, with reperfusion returning to pre-ischemic levels by day 10 post-ischemia compared with only ~30% reperfusion in control mice (P<0.05, n=8). This was associated with increased eNOS activity, measured by radioimmunoassay. In vitro angiogenesis in human umbilical vein endothelial cells (HUVECs) was determined using migration (% scratch wound closure) and tubule formation assays. HUVEC migration was increased in a concentration-dependent manner after β3AR stimulation, reaching 80±4% wound closure by 12 hours compared with 58±5% in controls (P<0.001, n=3). For tubule formation experiments, 2 x 104 cells were added to wells coated with matrigel and tubules formed over 4-12 hours. The formation of tubules was augmented by β3AR stimulation, with the maximal increase 70% higher than control (P<0.05, Student’s t-test). In both in vitro experiments, the NOS inhibitor, L-NAME (300 μmol/L) abolished the β3AR-stimulated proangiogenic effects. Experiments are ongoing to further elucidate the effects of β3AR stimulation on redox-signalling. Conclusion: β3AR stimulation increased angiogenesis in vitro and in vivo, likely by an NO-dependent mechanism. As β3AR agonists are approved in some countries for treating non-cardiovascular conditions they may prove a novel and imminently translatable therapy for diseases involving impaired angiogenesis, such as peripheral artery disease.

doi: 10.1016/j.freeradbiomed.2016.10.360 320 Differential Protein Acetylation Mediates Mitochondrial Localization and Pentameric Aggregation of SOD2 in the Murine Heart of SOD2Tg Chwen-Lih Chen1, Patrick T. Kang1, Liwen Zhang2, and Yeong-Renn Chen1 1 Northeast Ohio Medical University, Rootstown, USA, 2Ohio State University, Columbus, USA Cardiac-specific overexpression of SOD2 (mitochondrial manganese superoxide dismutase, mouse model of SOD2-tg) induces supernormal and physiologically hypertrophic phenotypes

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in the mouse heart. However, the reductive stress imposed by SOD2 overexpression also results in protein aggregation of SOD2 pentamer and differential acetylation of SOD2 in the compartments of mitochondrial matrix, mitochondrial inner membrane, and cytosol. We studied SOD2 acetylation in the matrix, submitochondrial particle (SMP), and cytosol from the SOD2-tg mouse heart. LC/MS/MS analysis revealed that K68, K75, K89, K114, K122, K130, K132, K134, and K154 residues of the cytosolic SOD2 were acetylated. LC/MS/MS analysis further indicated that K68, K122, and K130 of SOD2 in SMP and K68, K122, K 130, K202 of matrix SOD2 were acetylated. LC/MS/MS also detected a signaling peptide, 12QLAPVLGYLGSR23, in the SMP SOD2. The results implicated that mitochondrial localization of SOD2 resulted in specific deacetylation of K75, K89, K114, K132, K134, K154 residues, and site-specific acetylation of K202 residue in the matrix. In vitro acetylation of matrix SOD2 with acetic anhydride deaggregated pentameric SOD2, restored the profile of hyperacetylation observed in cytosolic SOD2, and decreased the enzymatic activity of matrix SOD2. As indicated by the 3D structure of SOD2, specific acetylation of K89, K134, and K154 detected in cytosolic SOD2 induces unfolding tertiary structure and breaking salt bridges for stabilizing the quaternary structure. Stable isotope labeling of both wild type and SOD2-tg matrix proteins followed by LC/MS analysis indicated significant up-regulation of stressinduced mitochondrial hsp70 (GRP75) and down-regulation of presequence protease (PREP), thus supporting increased reductive stress, compromised mitochondrial localization, and weakening correct folding of the nascent SOD2 in the SOD2-tg murine heart. In conclusion, hyperacetylation maintains the unfolded cytosolic SOD2 and mediates importation of SOD2 across the mitochondrial membrane. Activation of the matrix SOD2 is subsequently initiated by deacetylation that further induces SOD2 misfolding for pentameric aggregation.

doi: 10.1016/j.freeradbiomed.2016.10.361 321 DUSP4 Is the Key Protein Modulating Cardiovascular Function in Response to NAC Pre-Treatment Chun-An (Andy) Chen1 1 Ohio State University, Columbus, USA Our previous work on the redox activation of DUSP4 by NAC in endothelial cells demonstrated that DUSP4 is an antioxidant protein, critical in the modulation of MAPK signal pathways and eNOS expression by protecting against oxidant-induced stress. More recently, we demonstrated that DUSP4-/- mouse hearts suffer greater infarct than wild-type (WT) and have poor post-ischemic functional recovery when subjected to ischemia/reperfusion (I/R) injury. DUSP4 is an inducible nuclear phosphatase whose main function is to regulate MAPKs (ERK, JNK and p38) via dephosphorylation, and it has been demonstrated to be important for cardiovascular function. In this designed study, DUSP4-/- mouse is used to test that DUSP4 is the critical gene in response to NAC activation, which provides a beneficial effect against oxidant stress. WT or DUSP4-/- mice were first treated with NAC for two weeks, and subsequently hearts were isolated and subjected to 30 min ischemia and 30 min reperfusion. At the end of reperfusion period, the rate pressure product (RRP %) and coronary flow (CF %) were recorded to evaluate the functional recovery. The result indicated that WT hearts with NAC pre-treatment have a better functional recovery in both RPP (24.2% ± 7.7% vs 13.8% ± 2.9%) and CF (57.0% ±9.2% vs 36.8% ±2.8%) compared to non-treated WT hearts. However, DUSP4-/-hearts with NAC treatment exhibited poor functional recovery (lower in RPP (5.1% ± 1.0% s 3.0%

SfRBM / SFRRI 2016