The nitroxide 4-methoxy TEMPO inhibits neutrophil-stimulated kinase activation in H9c2 cardiomyocytes
Introduction
Acute myocardial ischaemia (AMI) is a common disease with serious mortality and morbidity outcomes and considerable societal costs. Occlusion of coronary vessels plays a crucial role in AMI and is usually a direct consequence of coronary artery disease (CAD). The risk factors for AMI are linked to CAD and include diabetes, hypertension and dyslipidaemia [1], [2]. Surviving AMI subjects are at risk of severe arrhythmias, persistent angina pectoris, secondary heart attack and chronic heart failure [3]. Treatment strategies aimed at restoring blood supply via reperfusion of the affected myocardium have shown positive therapeutic benefit [4]. However, ischaemic heart disease-related mortality remains a leading cause of death as up to 25% of patients that survive heart attack progress to chronic heart failure [5].
The reperfusion of occluded arteries initiates post-ischaemic events in the heart including promoting an inflammatory cascade and the recruitment of immune cells to the myocardium with neutrophils being prominent in the early phases after acute injury [6], [7]. Neutrophil recruitment to the affected myocardium following a heart attack initiates a reparative inflammatory process where matrix metalloproteinase (MMP)-mediated ventricular remodelling occurs. However, migratory neutrophils also release significant amounts of myeloperoxidase (MPO) and generate reactive oxygen species (ROS) upon activation [8].
Oxidants produced by MPO include the potent hypohalous acids such as hypochlorous acid (HOCl). Notably, both MPO and HOCl are associated with CAD [9], [10]. Hence, the presence of activated neutrophils in heart tissue post AMI has the potential to induce additional oxidative damage to the myocardium following ischaemia reperfusion injury. Muscle cells at the periphery of the primary infarct are within the “area-at-risk” zone, where neutrophils also congregate in response to chemotactic signals, and are particularly vulnerable to apoptotic cell death [11]. Circulating neutrophil counts after myocardial reperfusion are correlated with infarct size, myocardial tissue-level reperfusion, and left ventricle function [12]. Interestingly, patients in the highest tertile of circulating neutrophil count following an AMI show an increased risk of death or further acute events [13]. The preservation of myocardial viability is correlated with an improved prognosis and thus treatments aimed at modulating inflammation post AMI are potentially therapeutically important [14].
Nitroxides are widely documented for their antioxidant properties and have been identified as competitive substrates for MPO, effectively preventing HOCl formation in vitro [15]. Nitroxides arrest the MPO-H2O2-Cl- system through oxidation of MPO-compound I via one-electron transfer to the porphyrin π-cation radical yielding MPO compound II, a transition state for which nitroxides are poor substrates. This results in MPO-compound II accumulation and subsequent inhibition of the enzymic system, thereby abrogating HOCl production [16], [17]. In addition, nitroxides are highly cell-permeable with relatively low toxicity [18].
The aims of this work were to mimic oxidative damage to heart muscle by neutrophil-derived MPO/HOCl and to mitigate this pharmacologically using an in vitro co-culture system of H9c2 myocyte-like cells and activated neutrophils to monitor HOCl-mediated oxidative damage. Further the nitroxide, 4-methoxy TEMPO (MetT; compound [B]) was evaluated for its potential to modulate neutrophil-mediated oxidative damage in H9c2 cardiomyocytes (chemical structure shown together with the parental nitroxide TEMPOL; compound [A]).
Section snippets
Materials
Dulbecco's Modified Eagle's Medium/Ham's Nutrient Mixture F12 (DMEM/F12) was obtained from JRH Biosciences (Lenexa, KA). Unless otherwise indicated, all biochemicals were of the highest available purity and were obtained from Sigma Aldrich (Sydney, Australia).
Cell culture
The rat cardiomyoblast cell line, H9c2 was obtained from CellBank Australia (Sydney, Australia) and rat aortic smooth muscle cells (RASMC; used herein as a non-cardiac muscle cell) were from ATCC (Manassas, VA). Both cell types were
Cultured H9c2 cells express cardiac specific alpha-myosin
The rat H9c2 cardiomyoblast cell line exhibits cardiac muscle characteristics [40]. To confirm the phenotype of H9c2 cells in culture, the presence of cardiac-specific α-myosin (heavy chain) was determined [41]. Cultured H9c2 cells were passaged up to 8 times and assessed for α-myosin mRNA expression using previously published primers [36], [37], [38] (Fig. 1A). Cells cultured to a maximum of ∼80% confluency expressedα-myosin mRNA. By contrast, rat aortic smooth muscle cells (RASMC) showed
Discussion
Activation of neutrophils recruited to the affected myocardium following AMI may yield a secondary source of ROS, as neutrophil-MPO can facilitate HOCl formation resulting in a secondary bout of oxidative damage that potentially amplifies the initial injury [53], [54]. Therefore attenuating neutrophil-mediated ROS formation in the post AMI phase may be an effective strategy to alleviate oxidative damage in myocytes and to enhance the viability of the affected myocardium. Data from our
Conclusion and potential pathophysiological relevance
Here we show that MetT intervention mitigates neutrophil MPO-mediated damage to cultured cardiomyocytes. Cyclic nitroxides such as MetT are well tolerated when administered to rodents and show low toxicity [93] and our data suggests that MetT does not affect MMP activity, but rather appears to specifically modulate oxidative stress, and therefore is unlikely to impact negatively on factors important to wound healing and tissue remodelling. Irrespective of the mechanism of action of cyclic
Conflict of interest
The authors have no conflicts to declare.
Author contributions
B.C. – Performed a series of studies on the activity of protein tyrosine phosphatase (PTP) enzymes including investigations on thiol redox status in relation to PTP activity and was responsible for drafting the manuscript text.
G. J. – Performed Western blotting experimEents assessing MAPK activity and also immune fluorescent, flow cytometry and Elisa studies. G. J. worked with B. C. to develop the draft manuscript.
A. V. – Performed studies labelling cells with fluorescent transferrin and imaged
Sources of funding
The authors acknowledge funding from the Australian Research Council (DP0878559 and DP160102063 grants and NHMRC Project Grant 1125392 awarded to PKW).
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2021, Pharmacology and TherapeuticsCitation Excerpt :However, these nitroxides have also been shown to act as SOD mimetics, and are effective inhibitors of HOCl formation by activated neutrophils (Rees et al., 2009), indicating efficacy even in the presence of biological reductants including O2-•. Positive data have also been reported in an in vitro cardiomyocyte model of myocardial inflammation (Chami et al., 2017), in an animal model of colitis (Chami et al., 2020), a zebrafish model of tuberculosis (Black et al., 2019) and ex vivo in human plasma (Kajer et al., 2014). Furthermore, supplementation of animals with TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl radical) has been shown to stabilize atherosclerotic plaques by increasing collagen levels and decreasing lesion lipid content (C. H. Kim et al., 2015).
The cyclic nitroxide antioxidant 4-methoxy-TEMPO decreases mycobacterial burden in vivo through host and bacterial targets
2019, Free Radical Biology and MedicineCitation Excerpt :Neutrophils primarily kill bacteria through myeloperoxidase, an enzyme that produces potently microbicidal hypochlorous acid and could trigger macrophage necrosis during infection [8,42]. Recently, MetT was identified as a potent inhibitor of myeloperoxidase-induced production of hypochlorous acid in an in vitro neutrophil co-culture model of acute myocardial infarction [32]. However, using a loss of function mutant of neutrophil myeloperoxidase we show that myeloperoxidase is dispensable for control of mycobacterial infection by zebrafish embryos.
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2018, Archives of Biochemistry and BiophysicsCitation Excerpt :Protein tyrosine phosphatases (PTPs) are involved in a diverse range of cellular processes and regulate the total cellular phosphorylative status by regulating mitogen-acivated protein kinase (MAPK) activity, namely p38a and extracellular signal regulated kinase (ERK1/2). Sub-lethal doses of reagent HOCl (20–30 μM) have been employed to demonstrate HOCl-mediated activation of the MAPKs, (ERK1/2) and p38 in different cell types [80,81]. However, the co-administration of a MEK inhibitor, an upstream activator of ERK1/2, inhibits HOCl-mediated activation of ERK1/2, therefore ruling out a direct relationship between HOCl and ERK1/2 [81].
Nitroxides mitigate neutrophil-mediated damage to the myocardium after experimental myocardial infarction in rats
2020, International Journal of Molecular Sciences
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Authors contributed equally to the preparation of this manuscript.