M40403 prevents myocardial injury induced by acute hyperglycaemia in perfused rat heart

https://doi.org/10.1016/j.ejphar.2004.06.037Get rights and content

Abstract

M40403 is a low-molecular-weight, synthetic manganese-containing biscyclohexylpyridine superoxide dismutase mimetic (SODm) that removes superoxide anions (radical dotO2) without interfering with other reactive species known to be involved in cardiovascular alterations (e.g. nitric oxide [NO] and peroxynitrite [ONOO]). As such, M40403 represents an important pharmacological tool to dissect the roles of O2 in functional and biochemical cardiovascular alterations induced by perfusion of high glucose concentrations into the heart.

Perfusion of a high glucose concentration of glucose into the heart elicited important cardiovascular alterations characterized by QT interval prolongation, increase in coronary perfusion pressure (CPP), lipid peroxidation, decrease in MnSOD activity and DNA damage. All parameters of cardiovascular alteration were attenuated by M40403 (1–10 mg/l). Furthermore, perfusion of a high of glucose concentration induced a significant formation of nitrotyrosine as well as an activation of poly(adenosine diphosphate [ADP]-ribose) synthetase (PARS), as determined by immunohistochemical analysis of heart tissue. The extent of staining for nitrotyrosine and PARS was reduced by M40403.

These results clearly indicate that O2 plays a critical role in the development of the functional and biochemical cardiovascular alterations induced by perfusion of a high concentration of glucose into the heart. Therefore, synthetic enzymes of SOD, such as M40403, offer a novel therapeutic approach for the management of various cardiovascular diseases where these radicals have been postulated to play a role.

Introduction

Oxidative stress is an important component of diabetes and its complications (Ammar et al., 2000, Cakatay et al., 2000, Cameron et al., 1998, Cameron and Cotter, 1995, Cameron and Cotter, 1999, Gocmen et al., 2000, Haak et al., 2000, Ishi et al., 1998, Keegan et al., 1999, Obrosova et al., 2000, Pieper et al., 1993, Pieper, 2000, Pieper and Siebeneich, 1997). Cardiovascular disease is a major complication of diabetes, leading to mortality in diabetic patients (Stamler et al., 1993). In the last few years, it has been demonstrated that hyperglycaemia may be one of the causes of the cardiovascular alterations (e.g. microvascular diseases, coronary heart disease and peripheral arterial disease) associated with diabetes (The DCCT Research Group, 1993, Kuusisto et al., 1994). Therefore, hyperglycaemia may represent an important predictive risk factor for hypertension and myocardial infarction in patients with and/or without diabetes (Capes et al., 2000). In this regard, it is not surprising that an increase in the glucose level can induce arrhythmias with QT prolongation, fatal in the case of infarction, and increases coronary constriction (D'Amico et al., 2001). Recently, it has been demonstrated that treatment with antioxidants reduces the oxidative stress and vascular dysfunction associated with experimental diabetes (Cameron et al., 1993, Cameron et al., 1994, Cameron et al., 1998, Cameron and Cotter, 1999, Karasu et al., 1995, Keegan et al., 1999). Recent studies have clearly demonstrated that an increased production of reactive oxygen species is responsible for the hyperglycaemia-induced electrophysiological alterations (Cervello et al., 2002). However, none of the previous studies identified the particular radical(s) involved in the hyperglycaemia-induced electrophysiological alterations. This stems from the fact that selective antioxidants have not been available. In various pathological situations, the use of native superoxide dismutase (SOD) enzymes both pre-clinically and clinically has shed light on the importance of O2 in disease and, thus, the therapeutic potential of exogenous SOD enzymes (Flohe, 1988, Huber et al., 1980, Uematsu et al., 1994). However, the native SOD enzyme has not been evaluated in hyperglycaemia-induced electrophysiological alterations. Thus, the role of superoxide in this condition has not been defined. There are drawbacks or problematic issues associated with the use of native enzymes as therapeutic agents (e.g., solution instability, immunogenicity of non-human enzymes, bell-shaped dose–response curves, high susceptibility to proteolytic digestion) and as pharmacological tools (e.g., they do not penetrate cells or cross the blood–brain barrier, limiting the dismutation of superoxide to the extracellular space or compartment). To overcome the limitations associated with native enzyme therapy, we have developed a series of SOD mimetics that catalytically remove O2. M40403 is a prototypic example of a stable, low-molecular-weight, manganese-containing, non-peptidic molecule possessing the function and catalytic rate of native SOD enzymes, but with the advantage of being a much smaller molecule (Salvemini et al., 1999). An important property of these SOD mimetics is that they catalytically remove superoxide at a high rate without interacting with other biologically important reactive species including nitric oxide, peroxynitrite, hydrogen peroxide, oxygen or hydroxyl radicals (Riley et al., 1996, Riley et al., 1997). This property is not shared by other classes of SOD mimetics or scavengers, including several metalloporphyrins such as tetrakis-(N-ethyl-2-pyridyl)porphyrin and tetrakis-(benzoic acid)porphyrin, which interact with other reactive species, such as nitric oxide and peroxynitrite, that play important roles in inflammation (Patel and Day, 1999). Therefore, the purpose of our study was to evaluate whether interventions against O2 with M40403, a synthetic manganese-containing biscyclohexylpyridine SODm (Fig. 1), prevent the functional and biochemical cardiovascular alterations induced by perfusion of a high concentration of glucose into the heart. In particular, we investigated the effect of M40403 in isolated working rat hearts perfused with a high concentration of glucose on (i) QT interval prolongation, (ii) coronary perfusion pressure (CPP) increase, (iii) lipid peroxidation (malondialdehyde levels), (iv) superoxide dismutase (SOD) activity, (v) the nitration of tyrosine residues (an indicator of the formation of peroxynitrite) (by immunohistochemistry), (v) poly(adenosine diphosphate [ADP]-ribose) synthetase (PARS) activation and (vi) DNA damage (8 hydroxy-2′-deoxyguanosine formation).

Section snippets

Cell culture

Human umbilical vein endothelial cells (BioWhittaker, Walkersville, USA) were cultured in Endothelial Growth medium Bullet Kit w/2% FBS (BioWhittaker). Cells were cultured in 96-well plates (200 μl medium/well) or in 12-well plates (3 ml medium/well) until 90% confluence. Cells were exposed to hydrogen peroxide (10 mM for 2 h) or peroxynitrite (1 mM for 30 min), in the presence or absence of M40403 (10–1 μM).

Measurement of mitochondrial respiration

Cell respiration was assessed by the mitochondrial-dependent reduction of

Effects of high glucose on heart function

Heart rate (RR interval, 230±19 ms) and atrioventricular conduction time (PR interval, 59±9 ms) were similar for all groups.

In the hearts perfused with the control buffer solution, QT was 110±7 ms and remained steady throughout all the perfusion. d-Glucose (33.3 mmol/l) caused a significant prolongation of QT (202±19 ms, p<0.001 vs. control). The effect of high glucose was completely prevented by M40403 10 mg/l (QT, 107±11 ms), but unaffected by M40403 1 mg/l.

In hearts perfused with the control

Discussion

The present study demonstrates, for the first time, that the cardiovascular effects elicited by acute exposure of isolated rat hearts to high glucose could be prevented by infusion of M40403. In fact, the study shows that M40403, which is already reported to be beneficial in models of acute and chronic pathologies (Cuzzocrea et al., 2001a, Cuzzocrea et al., 2001b, Masini et al., 2002, Salvemini et al., 2001), reverses the cardiac QT interval prolongation and CPP increase induced by a high

Acknowledgement

This study was supported by a grant of MIUR, Italy.

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