Recombinant Human Matrix Metalloproteinase-2 Impairs Cardiovascular b-Adrenergic Responses Karina C. Ferraz 1 , Ozélia Sousa-Santos 2 , Evandro M. Neto-Neves 2 , Elen Rizzi 2 , Jaqueline J. Muniz 1 , Raquel F. Gerlach 3 and Jose E. Tanus-Santos 2 1 Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas, Campinas, Brazil, 2 Department of Pharmacology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil, and 3 Department of Morphology, Stomatology and Physiology, School of Dentistry of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil (Received 28 June 2012; Accepted 9 August 2012) Abstract: Growing evidence supports the involvement of matrix metalloproteinases (MMPs) in the pathogenesis of many cardio- vascular diseases. Particularly, imbalanced MMP-2 activity apparently plays a critical role in cardiovascular remodelling. While some studies have suggested that MMP-2 may affect the vascular tone and impair b-adrenoreceptor function, no previous study has examined the acute haemodynamic effects of MMP-2. We examined the effects of recombinant human MMP-2 (rhMMP-2) administered intravenously to anaesthetized lambs at baseline conditions and during b 1 -adrenergic cardiac stimulation with dobu- tamine. We used 26 anaesthetized male lambs in two study protocols. First, rhMMP-2 (220 ng/kg/min. over 60 min.) or vehicle was infused in the lambs, and no significant haemodynamic changes were found. Therefore, we infused dobutamine at 5 lg/kg/ min. i.v. (or saline) over 180 min. in lambs that had received the same rhMMP-2 infusion preceded by doxycycline i.v. at 10 mg/kg (or saline). Plasma and cardiac MMP-2 levels were assessed by gelatin zymography, and gelatinolytic activity was assessed by spectrofluorimetry. Dobutamine decreased systemic vascular resistance index, and this effect was attenuated by rhMMP-2 infusion. Moreover, dobutamine increased the cardiac index and left ventricular dP/dt max , and these effects were atten- uated by rhMMP-2. The previous administration of doxycycline blunted rhMMP-2-induced changes in dobutamine responses. While the infusion of rhMMP-2 did not increase plasma and cardiac MMP-2 levels, it increased cardiac gelatinolytic activity, and doxycycline blunted this effect. Our findings show that rhMMP-2 exerts no major haemodynamic effects in lambs. However, rhMMP-2 impairs the responses elicited by activation of b-adrenoreceptors. Growing evidence supports the involvement of a group of enzymes named matrix metalloproteinases (MMPs) in the pathogenesis of many disease conditions, including diseases affecting the cardiovascular system [1–4]. Particular attention has been paid to MMP-2 because imbalanced MMP-2 activity apparently plays a critical role in cardiovascular remodelling [5–7] and in other alterations of the cardiovascular system [8– 10]. However, recent studies are clearly showing that MMP-2 may have many other targets unrelated to the extracellular matrix, including intracellular substrates [11,12] and other mediators possibly affecting the vascular tone such as big- endothelin-1[13], calcitonin gene–related peptide [14] and adrenomedullin [15]. Importantly, activated MMP-2 has been shown to impair cardiac function possibly as a result of its activity targeting sarcomeric and cytoskeletal proteins such as troponin I, myosin light chain-1, a-actinin and titin [16–20]. Recent studies indicate that MMPs, including MMP-2, are involved in proteolytic cleavage of b 1 - and b 2 -adrenoreceptors [21]. Rodrigues et al. [22] demonstrated that the labelling den- sity of the extracellular domain of b 2 -adrenergic receptor in aortic endothelial cells from Wistar rats was reduced after treatment with plasma of spontaneously hypertensive rats with increased MMPs levels, thus indicating cleavage of b 2 -adren- ergic receptors. Supporting their findings, another study showed that MMP-2 is involved in the proteolysis of the extracellular domain of b 2 -adrenoreceptors in kidney from spontaneously hypertensive rats [23]. Furthermore, Hakalahti et al. [24] showed that GM6001 (a non-specific MMP inhibi- tor) prevented the cleavage of the N-terminus of the b 1 -adren- ergic receptor. While many studies implicate elevated MMP-2 levels in dis- ease conditions, no previous study has examined the acute haemodynamic effects of MMP-2. Moreover, no previous study has examined the acute effects of MMP-2 on cardiac function. In this study, we examined the effects of recombi- nant human MMP-2 (rhMMP-2) [25] administered intrave- nously to anaesthetized lambs. Moreover, we have suggested that rhMMP-2 could impair b 1 -adrenergic cardiac stimulation with dobutamine. Materials and Methods Expression of full-length recombinant human MMP-2. We expressed rhMMP-2 in Escherichia coli as previously described [25]. In addition, we tested the functionality of this rhMMP-2 using gelatin zymography and a fluorimetric assay as described below [25]. Animal model and haemodynamic measurements. The study complied with the guidelines of the Faculty of Medicine of Ribeirao Preto, Author for correspondence: Jose E. Tanus-Santos, Department of Phar- macology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Av. Bandeirantes, 3900, 14049-900 Ribeirao Preto, SP, Brazil (fax + 55 1636020220, e-mail tanus@fmrp.usp.br; tanussantos@yahoo.com). © 2012 The Authors Basic & Clinical Pharmacology & Toxicology © 2012 Nordic Pharmacological Society Basic & Clinical Pharmacology & Toxicology, 2013, 112, 103–109 Doi: 10.1111/bcpt.12001