Methylene blue alleviates endothelial dysfunction and reduces oxidative stress in aortas from diabetic rats.

Endothelial dysfunction and the related increase in reactive oxygen species (ROS) production are important events in the pathophysiology of diabetes mellitus (DM). Methylene blue (MB) has been systematically investigated for its protective effects against refractory hypotension and mitochondrial dysfunction. We have previously demonstrated that MB improved mitochondrial respiration and partially decreased oxidative stress in diabetic rat hearts. The present study was aimed to investigate whether MB modulates vascular function and ROS production in thoracic aortic rings isolated from rats with streptozotocin-induced DM (after 4 weeks of hyperglycemia). The effects of MB (0.1 µM, 30 min ex vivo incubation) on vascular reactivity in organ chamber (phenylephrine-induced contraction, acetylcholine-induced relaxation) and H2O2 production (assessed by ferrous iron xylenol orange oxidation assay) were investigated in vascular preparations with intact endothelium and after denudation. DM elicited a significant alteration of vascular function: increased contractility to phenylephrine, attenuation of acetylcholine-dependent relaxation, and augmented H2O2 generation. Ex vivo incubation with MB partially reversed all these changes (by approximately 70%) in vascular segments with intact endothelial layer (but not in denuded vessels). In conclusion, MB might be useful in alleviating endothelial dysfunction and mitigating endothelial oxidative stress, observations that clearly require further investigation in the setting of cardiometabolic disease.

Methylene blue improves mitochondrial respiration and decreases oxidative stress in a substrate-dependent manner in diabetic rat hearts.

Diabetic cardiomyopathy has been systematically associated with compromised mitochondrial energetics and increased generation of reactive oxygen species (ROS) that underlie its progression to heart failure. Methylene blue is a redox drug with reported protective effects mainly on brain mitochondria. The purpose of the present study was to characterize the effects of acute administration of methylene blue on mitochondrial respiration, H2O2 production, and calcium sensitivity in rat heart mitochondria isolated from healthy and 2 months (streptozotocin-induced) diabetic rats. Mitochondrial respiratory function was assessed by high-resolution respirometry. H2O2 production and calcium retention capacity were measured spectrofluorimetrically. The addition of methylene blue (0.1 µmol·L-1) elicited an increase in oxygen consumption of mitochondria energized with complex I and II substrates in both normal and diseased mitochondria. Interestingly, methylene blue elicited a significant increase in H2O2 release in the presence of complex I substrates (glutamate and malate), but had an opposite effect in mitochondria energized with complex II substrate (succinate). No changes in the calcium retention capacity of healthy or diabetic mitochondria were found in the presence of methylene blue. In conclusion, in cardiac mitochondria isolated from diabetic and nondiabetic rat hearts, methylene blue improved respiratory function and elicited a dichotomic, substrate-dependent effect on ROS production.

The effect of purinergic signaling via the P2Y11 receptor on vascular function in a rat model of acute inflammation.

There is a growing body of evidence pointing to the role of purinergic signaling in the development and progression of various conditions that have inflammation as a common pathogenetic denominator. The aim of the present study was to assess the involvement of P2Y11 purinergic receptors in the regulation of vascular function in aortic segments obtained using an experimental model of acute inflammation, the lipopolysaccharide (LPS, 8 mg/kg, i.p)-treated rats. Twelve hours after LPS administration, thoracic aortas were isolated and used for studies of vascular reactivity in the organ bath and for the measurement of reactive oxygen species (ROS) generation, respectively. LPS treatment significantly increased contractility to phenylephrine and attenuated the endothelium-dependent relaxation of the vascular segments in response to acetylcholine; an increased production of hydrogen peroxide (H2O2) was also recorded. The P2Y11 activator, NF546, decreased the LPS-induced aortic H2O2 release and partially normalized the vasomotor function, namely reduced contractility and improved relaxation. The effect was abolished by co-treatment with the P2Y11 inhibitor, NF340, and also after endothelium denudation. Importantly, NF546 did not elicit an antioxidant effect by acting as a H2O2 scavenger, suggesting that the beneficial outcome of this treatment on the vasculature is the consequence of P2Y11 stimulation. In conclusion, purinergic P2Y11 receptors stimulation improves vascular function and mitigates oxidative stress in the setting of acute systemic inflammation, revealing salutary effects and therapeutic potential in pathologies associated with endothelial dysfunction.

Acute inhibition of monoamine oxidase and ischemic preconditioning in isolated rat hearts: interference with postischemic functional recovery but no effect on infarct size reduction.

Monoamine oxidases (MAOs) have recently emerged as important mitochondrial sources of oxidative stress in the cardiovascular system. Generation of reactive oxygen species during the brief episodes of ischemic preconditioning (IPC) is responsible for the cardioprotection at reperfusion. The aim of this study was to assess the effects of two MAO inhibitors (clorgyline and pargyline) on the IPC-related protection in isolated rat hearts. Animals subjected to 30 min global ischemia and 120 min reperfusion were assigned to the following groups: (i) Control, no additional intervention; (ii) IPC, 3 cycles of 5 min ischemia and 5 min reperfusion before the index ischemia; (iii) IPC-clorgyline, IPC protocol bracketed for 5 min with clorgyline (50 µmol/L); (iv) IPC-pargyline, IPC protocol bracketed for 5 min with pargyline (0.5 mmol/L). The postischemic functional recovery was assessed by the left ventricular developed pressure (LVDP) and the indices of contractility (+dLVP/dt max) and relaxation (-dLVP/dt max). Infarct size (IS) was quantified by TTC staining. In both genders, IPC significantly improved functional recovery that was further enhanced in the presence of either clorgyline or pargyline. IS reduction was comparable among all the preconditioned groups, regardless of the presence of MAO inhibitors. In isolated rat hearts, acute inhibition of MAOs potentiates the IPC-induced postischemic functional recovery without interfering with the anti-necrotic protection.

BIOENERGETIC CHARACTERIZATION OF H9C2 CELLS USING THE EXTRACELLULAR FLUX ANALYZER.

UNLABELLED: The aim of the present work was to standardize the working methodology for assessing the bioenergetic profile of H9c2 cardiomyoblasts cells, with reference to the optimization of cell seeding number and the establishment of favorable concentrations for the classic modulators of mitochondrial respiratory function, in particular the one of a classical uncoupler, FCCP. MATERIAL AND METHODS: The extracellular flux analyzer (XF, Seahorse Bioscience) is a novel high-throughput instrument able to monitor the metabolism of living cells by simultaneously measuring mitochondrial respiration and glycolysis. The in vitro platform will be further used to better understand the pathophysiology and the unrecognized side effects of drugs currently used in the therapy of major cardiovascular diseases. CONCLUSIONS: In the long run, characterization of novel pharmacological agents' effects on other cell lines, including tumoral ones, will be also considered.

Ageing-induced decrease in cardiac mitochondrial function in healthy rats.

It is widely recognized that mitochondrial dysfunction is a key component of the multifactorial process of ageing. The effects of age on individual components of mitochondrial function vary across species and strains. In this study we investigated the oxygen consumption, the mitochondrial membrane potential (Δψ), the sensitivity of mitochondrial permeability transition pore (mPTP) to calcium overload, and the production of reactive oxygen species (ROS) in heart mitochondria isolated from old compared with adult healthy Sprague-Dawley rats. Respirometry studies and Δψ measurements were performed with an Oxygraph-2k equipped with a tetraphenylphosphonium electrode. ROS production and calcium retention capacity were measured spectrofluorimetrically. Our results show an important decline for all bioenergetic parameters for both complex I and complex II supported-respiration, a decreased Δψ in mitochondria energized with complex I substrates, and an increased mitochondrial ROS production in the old compared with the adult group. Mitochondrial sensitivity to Ca²âº-induced mPTP opening was also increased in the old compared with the adult animals. Moreover, the protective effect of cyclosporine A on mPTP opening was significantly reduced in the old group. We conclude that healthy ageing is associated with a decrease in heart mitochondria function in Sprague-Dawley rats.