Final Report Summary - HNO AND CVD (Role of Nitroxyl (HNO) in the cardio and cerebrovascular system)
Cardiac hypertrophy and ß-adrenergic desensitisation in mice in vivo were used as model of heart failure. In this study we tested whether HNO affects contractile force in normal and pathological ventricular myocytes (VMs) as well as in isolated hearts. VMs were isolated from mice either subjected to isoprenaline-infusion (ISO; 30 µg/g per day) or to vehicle (0.9 % NaCl) for 5 days. Sarcomere shortening and Ca2+ transients were simultaneously measured using the IonOptix system and force of contraction of isolated hearts was measured by a Langendorff perfusion system. HNO increased peak sarcomere shortening by ~200 % in a concentration-dependent manner (EC50 ~55 µM) and Ca2+ transient amplitude by ~120 %, suggesting effects on both myofilament Ca2+ sensitivity and sarcoplasmic reticulum (SR) Ca2+ cycling. The efficacy and potency did not differ between normal and chronic ISO VMs, despite the fact that the latter displayed a markedly diminished inotropic response to acute ß-adrenergic stimulation with ISO. Importantly, HNO did not affect diastolic Ca2+ or SR Ca2+ content, as assessed by rapid caffeine application. Furthermore, in isolated hearts, HNO increased force of contraction by 30 %, suggesting suitability in the whole organism in vivo. In conclusion, these data show that HNO increased contractile force in normal and ß-adrenergically desensitised VMs as well as in isolated mouse hearts. Thus, HNO may have potential application for the treatment of failing hearts.
Atherosclerosis was used as model of vascular disease. To examine the vasoactivity of HNO in isolated aortic rings and platelets, male wildtype (WT) and atherosclerotic apolipoprotein E-deficient (ApoE-/-) mice were used. HNO induced a concentration dependent relaxation (EC50 4.4 µM) of aortic rings isolated from WT and ApoE-/- mice. The concentration-response was markedly decreased in presence of excess GSH, an HNO scavenger, while the nitric oxide scavenger ODQ had no effect. Inhibitors of soluble guanylyl cyclase, calcitonin gene-related peptide receptor, and K+ channels (non-selective and voltage-dependent) each significantly impaired the vasodilator response to HNO. In contrast, inhibitors of adenylyl cyclase, ATP-sensitive K+ channel or high conductance Ca2+-activated K+ channel were ineffective. Furthermore, HNO significantly reduced contractile response and platelet aggregation mediated by thromboxane A2 mimetic U-46619 in a cGMP dependent manner. In conclusion, these results highlighted several mechanisms by which HNO counteracts vascular pathogenesis and promote HNO as a new class of therapeutics in vascular diseases.
Cerebral ischemia/reperfusion injury in vitro and in vivo was used to mimic cerebral disease. Ischemic stroke was induced in mice by middle cerebral artery occlusion (MCAO), and the effect of HNO on cortical infarct size, oxidative stress and blood pressure was investigated. The cortical infarct size was examined immediately and 2 days after MCAO using a six-point scale. As markers of oxidative stress we detected F2-isoprostanes 8-iso-PGF2a levels in urine by gas chromatography-mass spectrometry measurements and in serum ELISA measurements. The systolic blood pressure was measured non-invasively by tail-cuff plethysmography. We have found that HNO significantly increased the ischemic area in mice to 49.1 ± 9.1% of hemisphere as compared to 24.5 ± 5.7% of hemispheric volume in control mice injected with isotonic saline. After two days mice injected with HNO were severely neurologically impaired while control mice showed only a minimal neurological deficit (control 0.5 ± 0.3 n = 6; AS 2.2 ± 0.5). Urinary excretion of F2-isoprostanes 8-iso-PGF2a collected over a six h-period one day after MCAO induction, was significantly elevated in mice injected with HNO immediately prior to MCAO, compared with mice treated with saline (28.9 ± 2.7 ng/mg creatinine vs. 8.1 ± 5.3 ng/g creatinine; p <0.01). In plasma collected two days after stroke, the F2-isoprostane iPF2a-VI concentrations in HNO-treated mice showed a trend to higher levels compared to control mice, but this difference was not statistically significant (control 957 ± 309 pg/mL; AS 1369 ± 473 pg/mL; p = 0.34). Blood pressure measured 30 min after injection of HNO showed a decrease of approximately 30 mmHg, while it had no significantly changes immediately after HNO injection. These data showed that HNO exacerbates the neurological outcome and the infarct size in a mouse model of cerebral ischemia-reperfusion. We postulated that increased oxidative stress, aggravated neurotoxicity and reduced blood perfusion were possible explanations.
Due to its instability, the use of an HNO donor is necessary to characterise the chemistry and the biological functions of HNO. Many HNO donors are currently available but not all of them are amenable for in vivo or even in vitro studies. The most used HNO donors possess a relatively fast decomposition (t½=2-5 min) that results in a large, initial concentration of HNO that facilitates self-consumption, thus decreasing the overall amount of HNO available to react with biological targets. To overcome this limitation, longer-acting HNO donor compounds need to be developed. Thus, in this study we investigated an alternative HNO donor and we tested its effect on ventricular myocytes isolated from wildtype mouse hearts. By alkylation of diazeniumdiolated isopropylamine (IPA/NO), a known HNO donor, we synthesised iPrHN-N(O)=NO-CH2OAc (AcOM-IPA/NO) that resulted to be a stable derivative and easily purifiable by column chromatography. AcOM-IPA/NO hydrolysed to HNO an order of magnitude more slowly than IPA/NO at pH 7.4 and 37 oC, minimizing HNO self-consumption and enhancing trapping by biological targets such as metmyoglobin and glutathione. Consistent with the chemical trapping efficiency data, micromolar concentrations of AcOM-IPA/NO displayed significantly more potent sarcomere shortening effects relative to IPA/NO on ventricular myocytes isolated from wildtype mouse hearts. Thus, within these data we suggested AcOM-IPA/NO as a promising lead compound for the development of heart failure therapies.
Taken together, our findings provide a better understanding of the role of HNO in the cardio and cerebrovascular systems. In particular, we showed that HNO may have potential application for the treatment of cardiovascular diseases. However, the concentration of HNO known to be cardioprotective resulted to be detrimental in the nervous system. Therefore, the distinct effect of HNO on the nervous system must be taken into account when considering HNO as a therapeutic agent in the cardiovascular system.