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Myoglobin is presumably the most studied protein in biology. Its functional properties as a dioxygen storage and facilitator of dioxygen transport are widely acknowledged. Experimental evidence also implicates an essential role for myoglobin in the heart in regulating nitric oxide homeostasis. Under normoxia, oxygenated myoglobin can scavenge excessive nitric oxide, while under hypoxia, deoxygenated myoglobin can reduce nitrite, an oxidative product of nitric oxide, to bioactive nitric oxide. Myoglobin-driven nitrite reduction can protect the heart from ischemia and reperfusion injury. While horse and mouse myoglobin have been previously described to reduce nitrite under these conditions, a comparable activity has not been detected in human myoglobin. We here show that human myoglobin is a fully functional nitrite reductase. To study the role of human myoglobin for nitric oxide homeostasis we used repeated photometric wavelength scans and chemiluminescence based experiments. The results revealed that oxygenated human myoglobin reacts with nitrite-derived nitric oxide to form ferric myoglobin and that deoxygenated human myoglobin acts as a nitrite reductase in vitro and in situ. Rates of both nitric oxide scavenging and nitrite reduction were significantly higher in human compared to horse myoglobin. These data extend the existing knowledge about the functional properties of human myoglobin and are the basis for further translational studies towards the importance of myoglobin for nitric oxide metabolism in humans.

Background Nitrate and nitrite as oxidative derivates of nitric oxide have been suggested to both modulate and reflect exercise capacity in humans. The failure to increase endogenous nitrite has been related to decreased maximum exercise capacity, while a diet rich in nitrate improved exercise performance. The modulation of nitrate/nitrite levels has furthermore been related to an improvement of vascular functions. It remains unresolved to date whether baseline endogenous levels are associated with a superior vascular function and if this is also related to an improved exercise capacity. Objective To determine whether higher baseline nitrite levels are associated with a superior vascular function and whether this also relates to an advantage in exercise capacity as measured by lactate anaerobic threshold (LAT). Methods 12 male athletes with an average age of 25 ± 2 years, a body weight of 79 ± 4 kg, a height of 185 ± 5 cm and trained 5 ± 1 times per week (means ± SD) were enrolled in this study. Each participant reported twice to the testing facility ( n = 22 observations). Blood was drawn from the left cubital vein and the resulting plasma was used to determine the circulating levels of nitrate and nitrite via HPLC (ENO-20). 15 min later, vascular function was assessed on the contralateral arm (brachial artery) by measuring flow-mediated vasodilation (FMD) using a 12 MHz ultrasound probe. Participants were hereafter asked to complete a stepwise ergometric test starting at 100 W with increasing workloads (+50 W) every 5 min on a stationary bicycle ergometer (Lode Excalibur Sport). Blood was drawn from the ear lope and used to determine the levels of lactate at the end of each incremental steP Heart rate was measured continuously and the LAT (4 mM) in relation to heart rate was calculated using non-linear regression models. Results Baseline plasma nitrite levels correlated with LATs ( r = 0.65; p = 0.001, n = 22) and with endothelial function as assessed by FMD ( r = 0.71; p = 0.0002). Cor-relation coefficients from both testing days did not differ. Multiple linear regressions showed that base-line plasma nitrite level but not endothelial function was an independent predictor of exercise capacity. No such correlations were determined for plasma nitrate levels. Conclusion Higher baseline levels of nitrite are associated with a superior vascular function and lactate anerobic threshold. Disclosure M.T. was supported by a scholarship from the Deutsche Herzstiftung (German Heart Foundation), M.K. and T.R. were supported by grants from the German Research Foundation (DFG).

Background Methods for the assessment of vascular functions in mice are largely limited to ex vivo organ bath methods particularly the aortic ring assay. We aimed at establishing an easy to perform, sensitive, and valid method for the non-invasive measurement of vascular responses to pharmacological and physiological stimuli in vivo using laser Doppler perfusion imaging (LDPI). Methods Male C57Bl/6, 8–10 week old mice were anesthetized with isoflurane and tissue perfusion of the hindlimb was assessed by LDPI (Perimed, Stockholm, Sweden). The changes in tissue perfusion in response to vasodilators and vasoconstrictors was assessed by acute intraperitoneal (i.p) application of nitroglycerin, intravenous (i.v.) application of acethylcholine, and i.p application of epinephrine. Reactive hyperemic blood flow was induced by occluding the vessels of the hindlimb using an inflatable cuff until the perfusion signal reached the zero level. After 1–5 min of ischemia the cuff pressure was released. We determined intra-individual variability by measuring the same mouse on three consecutive days ( n = 4) and inter-individual variability by comparing the coefficient of variation (CV) of 11 mice. The effect of acute and chronic inhibition of nitric oxide synthase (NOS) or cyclooxygenase (COX) on the hyperemic response was achieved by administration of l -NNA or indometacin i.p or for 6 days in drinking water ad libitum, respectively. Results Application of nitroglycerin increased the perfusion of the hindlimb within approximately 100 s by 127% (191 ± 36 vs. 435 ± 23 perfusion units, PU), while epinephrine (209 ± 59 vs. 90 ± 18 PU:) resulted in a maximum decrease of 57% of the mean perfusion ( n = 5). I.v. administration of adenosine elevated the perfusion by 41% (224 ± 34 vs. 316 ± 43 PU). Reactive hyperemia progressively increased after 1, 3 and 5 min ischemia with the maximal response achieved after 5 min ischemia. Time to maximum reperfusion and the ratio of maximum/baseline perfusion values (perfusion reserve) showed the smallest variation, while the area under the perfusion curve exhibited a CV of 40–50%. Generally, intra-individual variability was lower as compared to inter-individual variability. Chronic oral treatment with l -NNA significantly prolonged the time to maximal perfusion (36 ± 5 vs. 77 ± 18 s), while acute NOS inhibition ( l -NNA i.P) reduced the perfusion reserve (2.5 ± 0.1 vs. 1.8 ± 0.0). In addition, chronic inhibition of COX resulted in a 16 s longer reperfusion (48 ± 5 vs. 64 ± 5 s) but had no effect on the other parameters. Conclusion Taken together, LDPI can be used as a valid and reproducible method for measuring vascular responses to physiological and pharmacological stimuli in the hindlimb of living mice. This may be a valuable experimental model that is similar to methodologies applied in humans enabling a translation of basic science findings. Disclosure Nothing to disclose.

Background Skeletal muscle regeneration is governed by complex cellular signaling processes. Proliferation of skeletal myoblasts is a key initial event in muscle regeneration and has been largely related to mammalian target of rapamycin (mTOR) signal transduction mechanisms. Although the precise underlying signaling remains incompletely understood, the role of nitric oxide (NO) in cell cycle regulation is well-appreciated. Nitrite, the oxidation product of NO, is regarded as a stable source for NO-like bioactivity in cases when the oxygen-dependent NO-synthases become dysfunctional. Whether nitrite itself has the potential to affect myoblast proliferation and metabolic activity under normoxic conditions with or without activation of the NO/cyclic guanosine monophosphate (cGMP) pathway is not known. Methods The influence of nitrite and NO on C 2 C 12 myoblast proliferation was observed in cultures grown in DMEM + 10% FCS at 37 °C/5% CO 2 , with or without sodium nitrite, NO-donor DETA-NONOate, NO-scavenger cPTIO, soluble guanylatecyclase (sGC) inhibitor ODQ, or mTOR inhibitor rapamycin. Neutral red staining, propodium iodide staining for cell cycle analysis, WST-8 viability assay, mTOR phosphorylation activity assay and intracellular cGMP measuerements were performed according to modified manufacturer’s protocols. Results We here show that nitrite increases proliferation and metabolic activity of murine cultured myoblasts dose-dependently in normoxia. This effect is not abolished by the NO scavenger 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and does not affect intracellular cGMP levels, implicating a cGMP-independent mechanism. Nitrite circumvents the rapamycin induced attenuation of myoblast proliferation and enhances mTOR phosphorylation activity. Conclusion Our results provide evidence for a novel potential physiological and therapeutic approach of nitrite in skeletal muscle regeneration processes under normoxia independent of NO and cGMP. Disclosure Nothing to disclose.

Purpose Revascularization is an adaptive repair mechanism that restores blood flow to undersupplied ischemic tissue. Nitric oxide (NO ) plays an important role in this process. Whether dietary nitrate, serially reduced to nitrite by commensal bacteria in the oral cavity and subsequently to NO , enhances ischemia-induced remodeling of the vascular network is not known. The present study was to assess the effects of dietary nitrate supplementation on ischemia-induced revascularization. Methods Mice were treated with either nitrate (1 g/L sodium nitrate in drinking water) or sodium chloride (control) for 14 days. At day 7, unilateral hindlimb surgery with excision of the left femoral artery was conducted. Blood flow was determined by laser doppler. Capillary density, myoblast apoptosis, mobilization of CD34+/Flk-1+, migration of bone-marrow derived CD31+/CD45−, plasma nitrite, nitrate and nitrosothiols as well as skeletal tissue cGMP levels were assessed. EGFP (enhanced green fluorescence protein) transgenic mice were used for bone marrow transplantation. Results Dietary nitrate supplementation leads to increased circulating nitrosothiol levels. Moreover nitrate supplementation enhances revascularization, increases mobilization of CD34+/Flk-1+ and migration of bone-marrow derived CD31+/CD45− cells to the site of ischemia, and attenuates apoptosis of potentially regenerative myoblasts in chronically ischemic tissue. The regenerative effects of nitrate treatment were abolished by eradicating nitrate-reducing bacteria in the oral cavity through an antiseptic mouthwash. Conclusions Chronic dietary nitrate supplementation may represent a novel nutrition-based strategy to enhance ischemia-induced revascularization. Disclosure Nothing to disclose.