The nitroxide 4-methoxy TEMPO inhibits neutrophil-stimulated kinase activation in H9c2 cardiomyocytes

https://doi.org/10.1016/j.abb.2017.07.001Get rights and content

Highlights

  • AMI is linked to neutrophil recruitment and activation that yields the damaging oxidant hypochlorous acid (HOCl) in the heart.

  • Exposure of H9c2 cells to neutrophils leads to P38 & ERK activation, inhibition of protein tyrosine phosphatases (PTP) and enhanced apoptosis.

  • Intervention with 4-methoxy-Tempo restored PTP & normalised MAPK activity and rescued H9c2 cell viability.

Abstract

After acute myocardial infarction (AMI), neutrophils are recruited to the affected myocardium. Hypochlorous acid (HOCl) produced by neutrophil myeloperoxidase (MPO) damages cardiomyocytes and potentially expands the primary infarct. Rat cardiomyocyte-like cells were incubated with isolated human neutrophils treated with chemical activators in the absence or presence of nitroxide 4-methoxy-Tempo (MetT; 25 μM) for 4, 6 or 24 h; studies with reagent HOCl served as positive control. Treating cardiomyocytes with activated neutrophils or reagent HOCl resulted in a marked increase in protein tyrosine chlorination and a decline in protein tyrosine phosphatase (PTP) activity. On balance our data also supported an increase in phosphorylation of MAPK p38 and ERK1/2 suggestive of an intracellular hyperphosphorylation status and this was accompanied by decreases in cell viability, as judged by assessing caspases-3/7 activity. For cells exposed to activated neutrophils receptor-mediated uptake of transferrin decreased although total matrix metalloproteinase (MMP) activity was unaffected. Addition of MetT ameliorated protein tyrosine chlorination, decreased MAPK activity and restored receptor-mediated transferrin uptake and PTP activity in cardiomyocytes. Overall, adverse effects of neutrophil-derived HOCl on cultured cardiomyocytes were ameliorated by MetT suggesting that nitroxides may be beneficial to inflammatory pathologies, where neutrophil recruitment/activation is a prominent and early feature.

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).

References (95)

  • J. Everse et al.

    Physical and catalytic properties of a peroxidase derived from cytochrome c

    Biochim. Biophys. Acta

    (2011)
  • T.H. Lu et al.

    Involvement of oxidative stress-mediated ERK1/2 and p38 activation regulated mitochondria-dependent apoptotic signals in methylmercury-induced neuronal cell injury

    Toxicol. Lett.

    (2011)
  • M. Lo Conte et al.

    The redox biochemistry of protein sulfenylation and sulfinylation

    J. Biol. Chem.

    (2013)
  • X. Fu et al.

    Hypochlorous acid oxygenates the cysteine switch domain of pro-matrilysin (MMP-7). A mechanism for matrix metalloproteinase activation and atherosclerotic plaque rupture by myeloperoxidase

    J. Biol. Chem.

    (2001)
  • J.L. Mehta et al.

    Neutrophils as potential participants in acute myocardial ischemia: relevance to reperfusion

    J. Am. Coll. Cardiol.

    (1988)
  • K. Lee et al.

    Inhibition of PTPs by H(2)O(2) regulates the activation of distinct MAPK pathways

    Free Radic. Biol. Med.

    (2002)
  • A. Samuni et al.

    A novel metal-free low molecular weight superoxide dismutase mimic

    J. Biol. Chem.

    (1988)
  • V.E. Kagan et al.

    Targeting nitroxides to mitochondria: location, location, location

    Free Rad. Biol. Med.

    (2007)
  • Y. Kinoshita et al.

    In vivo evaluation of novel nitroxyl radicals with reduction stability

    Free Radic. Biol. Med.

    (2010)
  • T.B. Kajer et al.

    Inhibition of myeloperoxidase- and neutrophil-mediated oxidant production by tetraethyl and tetramethyl nitroxides

    Free Rad. Biol. Med.

    (2014)
  • B.P. Soule et al.

    The chemistry and biology of nitroxide compounds

    Free Rad. Biol. Med.

    (2007)
  • N. Borregaard et al.

    Granules of the human neutrophilic polymorphonuclear leukocyte

    Blood

    (1997)
  • V. Fontaine et al.

    Involvement of the mural thrombus as a site of protease release and activation in human aortic aneurysms

    Am. J. Pathol.

    (2002)
  • N. Borregaard et al.

    Biosynthesis of granule proteins in normal human bone marrow cells. Gelatinase is a marker of terminal neutrophil differentiation

    Blood

    (1995)
  • Y. Wang et al.

    Myeloperoxidase inactivates TIMP-1 by oxidizing its N-terminal cysteine residue: an oxidative mechanism for regulating proteolysis during inflammation

    J. Biol. Chem.

    (2007)
  • J. Kalasz et al.

    Myeloperoxidase impairs the contractile function in isolated human cardiomyocytes

    Free Radic. Biol. Med.

    (2015)
  • E.K. Patterson et al.

    Carbon monoxide-releasing molecule 3 inhibits myeloperoxidase (MPO) and protects against MPO-induced vascular endothelial cell activation/dysfunction

    Free Radic. Biol. Med.

    (2014)
  • Q. Shao et al.

    Usefulness of neutrophil/lymphocyte ratio as a predictor of atrial fibrillation: a meta-analysis

    Arch. Med. Res.

    (2015)
  • C.H. Hennekens

    Increasing burden of cardiovascular disease: current knowledge and future directions for research on risk factors

    Circulation

    (1998)
  • L. Welin et al.

    Insulin resistance and other risk factors for coronary heart disease in elderly men. The Study of Men Born in 1913 and 1923

    Euro. J Cardiovasc. Prev. Rehab Off. J. Eur. Soc. Cardiol. Work. Groups Epidemiol. Prev. Cardiac Rehabil. Exerc. Physiol.

    (2003)
  • J. Marrugat et al.

    Mortality differences between men and women following first myocardial infarction. RESCATE Investigators. Recursos Empleados en el Syndrome Coronario Agudo y Tiempo de Espera

    J. Am. Med. Assoc.

    (1998)
  • J. Neuzil et al.

    Oxidative stress in myocardial ischaemia reperfusion injury: a renewed focus on a long-standing area of heart research

    Redox Rep.

    (2005)
  • P.S. Jhund et al.

    Heart failure after acute myocardial infarction: a lost battle in the war on heart failure?

    Circulation

    (2008)
  • B.F. Mervyn et al.

    Pathogenesis and modification of myocardial stunning and reperfusion injury

  • N.G. Frangogiannis

    Regulation of the inflammatory response in cardiac repair

    Circ. Res.

    (2012)
  • L.R. DeChatelet et al.

    An isotopic assay for NADPH oxidase activity and some characteristics of the enzyme from human polymorphonuclear leukocytes

    J. Clin. Invest

    (1975)
  • R. Zhang et al.

    Association between myeloperoxidase levels and risk of coronary artery disease

    J. Am. Med. Assoc.

    (2001)
  • R.M. Roman et al.

    Prognostic value of myeloperoxidase in coronary artery disease: comparison of unstable and stable angina patients

    Coron. Artery Dis.

    (2010)
  • M. Cavalera et al.

    Targeting the chemokines in cardiac repair

    Curr. Pharm. Design

    (2014)
  • K. Shinozaki et al.

    Significance of neutrophil counts after reperfusion therapy in patients with a first anterior wall acute myocardial infarction

    Circ. J.

    (2005)
  • O. Augusto et al.

    Cyclic nitroxides inhibit the toxicity of nitric oxide-derived oxidants: mechanisms and implications

    An. Acad. Bras. Cien.

    (2008)
  • M.D. Rees et al.

    Inhibition of myeloperoxidase-mediated hypochlorous acid production by nitroxides

    Biochem. J.

    (2009)
  • B.P. Soule et al.

    Therapeutic and clinical applications of nitroxide compounds

    Antiox. Redox Sig

    (2007)
  • J.J. Edelman et al.

    Off-pump coronary artery bypass surgery induces prolonged alterations to host neutrophil physiology

    Shock

    (2013)
  • L.G. Ribeiro et al.

    Quantification of hyperaemia bordering ischaemic myocardium in experimental myocardial infarction

    Cardiovasc. Res.

    (1980)
  • T. Sato et al.

    Molecular mechanisms of N-formyl-methionyl-leucyl-phenylalanine-induced superoxide generation and degranulation in mouse neutrophils: phospholipase D is dispensable

    Mol. Cell. Biol.

    (2013)
  • J.T. O'Flaherty et al.

    Neutrophil degranulation responses to combinations of arachidonate metabolites and platelet-activating factor

    Res. Comm. Chem. Pathol. Pharmacol.

    (1984)
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