Original article
Acetaminophen attenuates peroxynitrite-activated matrix metalloproteinase-2-mediated troponin I cleavage in the isolated guinea pig myocardium

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Abstract

The peroxynitrite-mediated activation of matrix metalloproteinase-2 (MMP-2) and subsequent cleavage of troponin I (TnI) in ventricular myocytes is a detrimental effect of ischemia/reperfusion injury. We hypothesized that acetaminophen, an effective antioxidant against peroxynitrite, would attenuate activation of MMP-2 and improve cardiac mechanical function. Isolated, perfused guinea pig hearts (Langendorff) were treated with either acetaminophen [0.35 mmol/l] or its vehicle and administered a bolus injection of peroxynitrite (6 μM) after reaching steady state function. Hemodynamic, metabolic, and mechanical effects were recorded, and coronary effluent concentrates or supernatant from heart homogenates were subjected to Western blotting and gelatin zymography. Hemodynamic and metabolic data showed no difference between acetaminophen- and vehicle-treated hearts. Mechanical data revealed that treatment with acetaminophen preserved contractile function (particularly diastolic function) after peroxynitrite administration. For example, 5 min after administration of peroxynitrite percent baseline –dP/dtmax was 10 ± 3% and –4 ± 7% (P < 0.05) in acetaminophen- and vehicle-treated hearts, respectively. Western blotting and gel zymography revealed higher 72 kDa (pro-MMP-2) proteolytic activity in heart homogenates of vehicle-treated versus acetaminophen-treated hearts. In addition, Western blotting of heart homogenates showed increased degradative products of TnI in vehicle-treated versus acetaminophen-treated hearts. We conclude that acetaminophen is cardioprotective, at least in part, by attenuating peroxynitrite-activated, MMP-2-mediated cleavage of TnI.

Introduction

In recent studies, our laboratory has provided evidence that acetaminophen is cardioprotective during both ischemia/reperfusion and hypoxia/reoxygenation in the isolated, perfused guinea pig heart [1], [2], [3]. Although this effect has been described, mechanistic data are lacking. The cardioprotective property of acetaminophen has been attributed to its antioxidant nature, as it retains the ability to reduce native peroxynitrite (ONOO) in the coronary vasculature [1], [3].

Although ONOO itself is damaging to myocardial and endothelial tissue, it has also been shown to activate mediators of cellular damage (e.g. the matrix metalloproteinases) [4], [5], [6], [7]. Recent studies have shown that the intracellular action of matrix metalloproteinase-2 (MMP-2) is responsible, at least in part, for the injury observed from acute release of ONOO following myocardial ischemia and reperfusion [8], [9]. Indeed, agents known to inhibit MMP-2, such as o-phenanthroline, can minimize damage caused by ONOO [8]. Activation of myocardial MMP-2 causes increased proteolysis of troponin-I (TnI), and results in a decrease in overall cardiac mechanical function [9], [10].

It is conceivable that antioxidants, such as acetaminophen, can attenuate the activation of intracellular MMP-2 and subsequent TnI cleavage by reducing circulating concentrations of ONOO. We therefore hypothesized that acetaminophen can attenuate the activation of myocardial MMP-2 through reduction of ONOO, and that this attenuation would inhibit cleavage of myocardial TnI.

Section snippets

Perfusate and perfusion

Unless otherwise stated, all reagents and biochemicals were obtained from Sigma-Aldrich, St. Louis, MO, USA. Hartley strain male guinea pigs (375 ± 25 g) were purchased from Charles River Laboratories (Wilmington, MA, USA), and were allowed to acclimate for a minimum of three days before being brought to the laboratory. In accordance with National Institutes of Health/United States Department of Agriculture guidelines and after IACUC review and approval, animals were anesthetized. A bilateral

Metabolic and vascular properties

There were no significant differences in the metabolic function of acetaminophen- and vehicle-treated hearts before or after administration of ONOO (Table 1). Arterial and venous O2 and CO2 levels were consistent throughout the duration of the experiment, and ONOO administration had no significant effect on pH (Table 1). Both base excess and calculated MVO2 also remained within values expected for this experimental setting. No significant differences were noted in vascular variables between

Discussion

Cleavage of cardiac TnI is a detrimental event arising from various causes and ultimately resulting in decreased Ca2+ responsiveness of myofilaments and contractile dysfunction [18], [19], [20]. Clinical application of the cleavage of TnI remains controversial, but degradation of TnI has been firmly demonstrated in the pathogenesis of myocardial stunning following ischemia/reperfusion or hypoxia/reoxygenation [18], [19], [21], [22], [23], [24]. The data presented above reveal that acetaminophen

Conclusions

Baseline levels of the activity of MMP-2 were similar in acetaminophen- and vehicle-treated hearts, and the administration of ONOO resulted in increased proteolytic activity of MMP-2 in vehicle- but not acetaminophen-treated hearts. The increase in proteolytic activity in vehicle-treated hearts was concomitant with cleavage of myocardial TnI; similar cleavage of TnI was not observed in acetaminophen-treated hearts. Simultaneously, diastolic function (mediated in large part by TnI) was

Acknowledgments

The authors wish to thank David Denhardt, Ph.D. for his indispensable technical assistance with all aspects of gelatin zymography and Western blotting. In addition, we wish to thank Erik Charych, Ph.D. for his technical assistance in homogenizations, Bonnie Firestein, Ph.D. for her generous donation of both time and materials, and Chingas Kemps for his technical assistance with software management. This work was funded in part by grants from Johnson & Johnson COSAT (New Brunswick, NJ), McNeil

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  • Cited by (0)

    Funded by McNeil Consumer and Specialty Pharmaceuticals (Fort Washington, PA), Johnson & Johnson, COSAT (New Brunswick, NJ), and the American Heart Association, Heritage Affiliate (Grant # 0555801T)

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