TGF-₁ attenuates myocardial ischemia-reperfusion injury via inhibition of upregulation of MMP-1

¹ Departments of Internal Medicine and Physiology, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, Arkansas 72205-7199; ² Department of Surgical Sciences, University of Uppsala, Uppsala 75237, Sweden

	ABSTRACT

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Ischemia-reperfusion (I/R) is thought to upregulate the expression and activity of matrix metalloproteinases (MMPs), which regulate myocardial and vascular remodeling. Previous studies have shown that transforming growth factor-₁ (TGF-₁) can attenuate myocardial injury induced by I/R. TGF-₁ is also reported to suppress the release of MMPs. To study the modulation of MMP-1 by TGF-₁ in I/R myocardium, Sprague-Dawley rats were given saline and subjected to 1 h of myocardial ischemia [total left coronary artery (LCA) ligation] followed by 1 h of reperfusion (n = 9). Parallel groups of rats were pretreated with recombinant TGF-₁ (rTGF-₁, 1 mg/rat, n = 9) before reperfusion or exposure to sham I/R (control group). I/R caused myocardial necrosis and dysfunction, indicated by decreased first derivative of left ventricular pressure, mean arterial blood pressure, and heart rate (all P < 0.01 vs. sham-operated control group). Simultaneously, I/R upregulated MMP-1 (P < 0.01). Treatment of rats with rTGF-₁ reduced the extent of myocardial necrosis and dysfunction despite I/R (all P < 0.01). rTGF-₁ treatment also inhibited the upregulation of MMP-1 in the I/R myocardium (P < 0.05). To determine the direct effect of MMP-1 on the myocardium, isolated adult rat myocytes were treated with active MMP-1, which caused injury and death of cultured myocytes, measured as lactate dehydrogenase release and trypan blue staining, in a dose- and time-dependent manner (P < 0.05). Pretreatment with PD-166793, a specific MMP inhibitor, attenuated myocardial injury and death induced by active MMP-1. The present study for the first time shows that MMP-1 can directly cause myocyte injury or death and that attenuation of myocardial I/R injury by TGF-₁ may, at least partly, be mediated by the inhibition of upregulation of MMP-1.

metalloproteinases; myocardium; transforming growth factor-₁

	INTRODUCTION

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MATRIX METALLOPROTEINASES (MMPs) are a family of zinc endopeptidases that share similar structure. Almost all cell types in ischemic myocardium, such as leukocytes, vascular endothelial cells, smooth muscle cells, fibroblasts, and myocytes, can synthesize and secrete MMPs, which degrade the extracellular matrix (ECM) (5, 7). In the early stages of ischemia-reperfusion, MMPs released from leukocytes degrade vascular ECM, breakdown basement membrane, increase vascular permeability, and enhance leukocyte migration outside the vascular lumen (5). MMPs secreted from the inflammatory cells and myocardial cells are also associated with myocardial rupture and apoptosis (13, 38). In the later stages of ischemia-reperfusion injury, release of MMPs is thought to relate to ventricular dilatation, aneurysm formation, and heart failure (5). Experimental studies suggest that inhibition of synthesis and activity of MMPs can protect the myocardium from the adverse effects of ischemia-reperfusion and significantly improve cardiac function (8, 26, 27).

Transforming growth factor-₁ (TGF-₁), a multifunctional polypeptide, is believed to influence cardiac development and function (25). Data from this and others' laboratories (24, 25) have shown that ischemia-reperfusion results in an increase in latent TGF-₁ levels but a decrease in its active form. Autoinduction of TGF-₁ expression as well as supplementation with exogenous TGF-₁ can protect the heart from ischemia-reperfusion injury (19, 20). Cardioprotection by TGF-₁ has been attributed to the inhibition of tumor necrosis factor- (TNF-) release, improvement of endothelium-dependent relaxation, and prevention of reactive oxygen species generation (18, 20); however, the precise mechanism is still unclear.

Increasing evidence suggests that TGF-₁ suppresses the expression of MMP-9 and -12 in monocytes and macrophages induced by cytokines, such as interleukin-1 (IL-1) and TNF- (11, 35). However, the modulation of MMPs by TGF-₁ in the myocardium in relation to ischemia-reperfusion has not been defined. The present study was designed to examine the direct effect of MMP-1 on cultured cardiomyocytes and the modulation of the expression of MMP-1 in myocardium subjected to ischemia-reperfusion by TGF-₁.

	MATERIALS AND METHODS

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Studies on Expression of MMP-1 in Ischemia-Reperfusion Myocardium by TGF-₁

Animals. Male Sprague-Dawley rats weighing 250-300 g were given saline and exposed to myocardial ischemia-reperfusion (n = 9). Parallel groups of rats were used as sham-operated controls (n = 8) or treated intravenously with human recombinant TGF-₁ (rTGF-₁, Sigma, 1 mg/rat, n = 9) in 0.5 ml of saline before reperfusion. The studies were approved by the animal protection committee of the University of Arkansas for Medical Sciences.

Myocardial ischemia-reperfusion injury. Rats were anesthetized, intubated, and mechanically ventilated on a positive pressure respirator with room air. Body temperature was maintained at 36-37°C with a heating blanket. A catheter was placed into the left ventricle via the right carotid artery to monitor positive and negative first derivatives of LV pressure (±dP/dt). Arterial pressure was monitored by placing a catheter in the right femoral artery. Heart rate was recorded by surface ECG. A left thoracotomy was performed via the fifth intercostal space to expose the heart. The main left coronary artery (LCA) was ligated 2-3 mm proximal to the origin of first diagonal branch with a 5-0 silk suture. Successful ligation was indicated by elevation of the ST segment on the ECG and by cyanosis of the anterior wall of the left ventricle. Hearts of rats in both untreated and TGF-₁-treated groups were subjected to 60 min of ischemia followed by 1 h of reperfusion (removal of the ligature). The sham-operated control group was subjected to thoracotomy and passage of silk ligature around LCA without ligation.

After completion of the experiment, half of the hearts in each group were used to measure infarct size, and the other half were saved to determine expression of TGF-₁ and MMP-1.

Determination of infarct size. At the end of in situ studies, hearts were quickly removed and mounted on Langendorff apparatus and then flushed with saline for 60 s. The LCA was reoccluded with a snare, and Evans blue dye was infused into the perfusate to mark the area at risk. The heart was then frozen and cut into 2-mm transverse slices. The slices were incubated in 1% triphenyl tetrazolium chloride (TTC) in 0.2 M PBS buffer (pH 7.4) for 5 min (36). The area of infarcted tissue (TTC-negative tissue) and the area at risk were determined by planimetry.

Determination of MMP-1 protein in myocardium. Method for the Western blot has been described previously (3, 4). The lysate protein from myocardial tissues (20 µg/lane) was separated by 10% SDS-PAGE. The primary monocolonal antibody to MMP-1 (Calbiochem; San Diego, CA) was used at 1:1,000 dilution. The bands were detected with the enhanced chemiluminescence (ECL) system, and relative intensity of bands of interest was analyzed.

Determination of MMP-1 mRNA in myocardium. The methodology for mRNA determination has been described earlier (3, 4). Briefly, 1 µg of total RNA was reverse transcripted with oligo-dT (Promega) and M-MLV reverse transcriptase (Promega) at 37°C for 1 h. RT material (1.5 µl) was amplified with Taq DNA polymerase (Promega) by using a primer pair specific to MMP-1 [forward primer: 5'-TTGTTGCTGCCCATGAGCTT-3'; reverse primer: 5'-ACTTTGTCGCCAATTCCAGG-3' (17)]. PCR product was 639 base pairs. For PCR, 30 cycles were used at 95°C for 45 s, 60°C for 45 s, and 72°C for 2 min. The RT-PCR amplified samples were visualized on 1.2% agarose gels by using ethidium bromide. A rat -actin primer pair was used as the control.

Studies on Direct Effect of MMP-1 on Cardiomyocytes

Myocyte isolation and culture and treatment with MMP-1 and MMP inhibitor. Calcium-tolerant myocytes were obtained from adult rat hearts as detailed earlier (4, 23). All procedures were carried out under aseptic conditions. Synthetic MMP-1 (Calbiochem) was activated with 1 mM of p-aminophenophenylmercuric acetate for 2 h at 37°C. Activated MMP-1 was stored in buffer containing (in mM) 50 Tris, 5 CaCl₂, and 200 NaCl. To examine the direct effect of MMP-1 on myocytes, cultured myocytes were incubated with MMP-1 (1, 3, and 10 nM) for 24 h and with 10 nM MMP-1 for 6, 12, and 24 h. In parallel, myocytes were pretreated with 1 µM of PD-166793 (a specific inhibitor of MMPs, Pfizer Global Research & Development; Ann Arbor, MI) for 1 h before incubation with 10 nM of MMP-1 for 24 h. At the end of the experiment, culture media were collected for determination of lactate dehydrogenase (LDH), and myocytes were harvested for determining cell death.

Determination of LDH in culture medium. A spectrophotometric method based on the oxidation of lactate (Sigma) was used to measure LDH release. LDH activity was expressed as units per milliliter of medium (4, 23).

Determination of cell death. Cell death was determined by the uptake of trypan blue by the cultured myocytes. The harvested cultured myocytes were incubated in 0.1% trypan blue for a few minutes and were observed under a light microscope. Dead cells, but not the viable myocytes, are permeable to trypan blue. At least 100 myocytes were counted in each field, and the percentage of viable cells was calculated.

Statistical analysis. All data are expressed as means ± SD of four separate experiments. Means of different groups were compared with ANOVA, followed by Newman-Keuls test for paired and unpaired observations. A P value <0.05 was considered significant.

	RESULTS

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Effect of TGF-₁ on Cardiac Dynamics

In the sham-operated control group of rat hearts (n = 8), there were only minimal (~5%) changes in dP/dt, mean arterial pressure, and heart rate. Rats given saline and subjected to 60 min of ischemia followed by 1 h of reperfusion showed marked cardiac dysfunction, indicated by the significant reduction of dP/dt, mean arterial pressure, and heart rate (all P < 0.01 vs. preischemia values). Treatment of rats with rTGF-₁ markedly attenuated the ischemia-reperfusion-induced cardiac dysfunction, indicated by preservation of dP/dt, mean arterial pressure, and heart rate (all P < 0.01 vs. untreated controls) (Fig. 1A).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   A: summary of effect of transforming growth factor-1 (TGF-1) on cardiac dynamics. There were only minimal (~5-10%) changes in first derivative of left ventricular (LV) pressure (dP/dt), mean arterial pressure (MAP), and heart rate (HR) in the sham-operated control group (n = 8). Ischemia-reperfusion (I/R) in untreated controls resulted in a marked decrease in dP/dt, MAP and HR (n = 9, all P < 0.01). Treatment of rats with recombinant TGF-1 (rTGF-1) markedly attenuated the I/R-induced cardiac dysfunction (n = 9, all P < 0.01, vs. untreated group). B: effect of TGF-1 on infarct size. I/R caused extensive myocardial necrosis. Treatment of rats with rTGF-1 reduced infarct size (n = 5, P < 0.01).

Effect of TGF-₁ on Infarct Size

As shown in Fig. 1B, total ischemia for 1 h followed by 60 min of reperfusion caused extensive myocardial necrosis. Treatment of rats with rTGF-₁ markedly reduced the infarct size as a percentage of thearea at risk (n = 5, P < 0.01 vs. untreated group).

TGF-₁ Expression in Rat Hearts

Consistent with our previous observations (23), ischemia-reperfusion markedly increased TGF-₁ expression in myocardial tissues (n = 4, P < 0.01 vs. sham-operated control group). Treatment with exogenous rTGF-₁ inhibited ischemia-reperfusion-induced TGF-₁ upregulation in the myocardium (n = 4, P < 0.01 vs. untreated group) (Fig. 2).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   TGF-1 expression in rat hearts. I/R markedly increased TGF-1 protein expression in myocardial tissues. Administration of exogenous rTGF-1 inhibited I/R-induced TGF-1 protein expression in myocardium. A: representative experiments (2 from each group); B: summary (means ± SD) of 4 separate experiments.

Effect of TGF-₁ on MMP-1 Expression

As shown in Fig. 3A, ischemia-reperfusion resulted in an increase in the expression of both active (43 kDa) and latent (52 kDa) forms of MMP-1 in the myocardium (n = 4, P < 0.01 vs. sham-operated control group). Treatment of rats with rTGF-₁ decreased the ischemia-reperfusion-induced MMP-1 (both active and latent) expression in rat hearts (n = 4, all P < 0.05 vs. untreated group). Figure 3B shows the MMP-1 gene expression in myocardial tissues. These results are consistent with the results of MMP-1 protein expression.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   A: matrix metalloproteinases-1 (MMP-1) expression in rat hearts. I/R increased both active and latent MMP-1 protein expression in myocardial tissues. Treatment of rats with rTGF-1 markedly decreased the I/R-induced MMP-1 expression (both active and latent). Top, representative experiments (2 from each group); bottom, summary (means ± SD) of 4 separate experiments. B: consistent with the Western blot data (left), I/R also induced MMP-1 mRNA expression in myocardial tissues. Treatment of rats with rTGF-1 markedly decreased I/R-induced MMP-1 mRNA expression. Top, representative experiments (2 from each group); bottom, summary (means ± SD) of 4 separate experiments.

Direct Effect of MMP-1 on Cultured Myocytes

As shown in Fig. 4, exogenous active MMP-1 caused myocyte injury in a dose- and time-dependent manner (n = 5, P < 0.05), as indicated by LDH release in the culture media. Exogenous MMP-1 also caused death of myocytes, as determined by trypan blue dye (n = 5, P < 0.01). Pretreatment of myocytes with PD-166793 markedly attenuated myocyte injury and death induced by exogenous MMP-1 (n = 5, P < 0.05 vs. MMP-1 alone).

View larger version (29K): [in this window] [in a new window]   Fig. 4.   A: effect of MMP-1 on lactate dehydrogenase (LDH) release. Exogenous active MMP-1 caused a marked increase in LDH release in the culture media in a dose- and time-dependent manner. Pretreatment of cultured myocytes with PD-166793 (1 µM) attenuated LDH release in response to MMP-1 (10 nM). Data from 5 separate experiments are expressed as means ± SD. B: effect of MMP-1 on death of cardiomyocytes. Consistent with the result of LDH release in the culture media, exogenous MMP-1 (10 nM) markedly increased the number of dead myocytes. Pretreatment of cultured myocytes with PD-166793 (1 µM) reduced the number of dead despite treatment with MMP-1. Data from 5 separate experiments are in means ± SD.

	DISCUSSION

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We and others (4, 18-20, 24) have shown the cardioprotective effect of TGF-₁ during ischemia-reperfusion. We confirm this phenomenon in the present in vivo study. In addition, we provide novel insights into the mechanism of the cardioprotective effect of TGF-₁.

TGF-₁ and Myocardial Ischemia-Reperfusion Injury

There is emerging clinical evidence for a link between TGF-₁ gene polymorphism, which affects serum TGF-₁ levels, and the presence of coronary artery disease (15, 39). Serum TGF-₁ levels are generally decreased in patients with coronary atherosclerosis and correlate inversely with the presence of myocardial ischemia, implicating arelative lack of active TGF-₁ in atherogenesis (1, 31, 33, 34). Recent studies (1, 33) also indicate that the plasma concentration of TGF-₁ may have a clinical prognostic significance in patients with coronary artery disease. Experimental studies (12, 24, 40) show that TGF-₁ expression is upregulated in the ischemic-reperfused tissues. Studies from Mehta's laboratory (24) have demonstrated that both TGF-₁ mRNA and latent TGF-₁ levels are elevated, but active TGF-₁ is reduced in the rat myocardium during ischemia-reperfusion, which indicates that there may be a defect in the conversion of latent TGF-₁ to its active form during ischemia.

Although traditionally thought of as a profibrotic factor, TGF-₁ is now well recognized to have potent tissue protective effects, particularly during ischemia-reperfusion (4, 18, 20). The cardioprotective effect of exogenous TGF-₁ is associated with the preservation of endothelium-dependent relaxation, prevention of free radical generation, and a decrease in TNF- release (19, 20). We (4) have shown that TGF-₁ modulates the expression of nitric oxide synthase by modulating the protein kinase B (PKB/Akt) pathway. Baxter et al. (2) have suggested that TGF-₁ can modulate the activation of p42/44 mitogen-activated protein kinase.

MMPs and Cell Injury

MMPs play numerous physiological roles in wound healing and angiogenesis. When dysregulated, they also participate in pathological processes such as plaque rupture in atherosclerosis and remodeling of blood vessels and hearts after ischemia (6, 10, 30). Clinical studies (14, 16) show increased plasma levels of MMPs in patients with acute myocardial infarction. The elevated MMP levels may contribute to cardiac remodeling. There is also evidence that MMPs may be activated in the very early stages of myocardial ischemia-reperfusion injury (5, 22). MMPs released from leukocytes can degrade vascular ECM, breakdown basement membrane, increase vascular permeability, and enhance leukocyte migration outside the vascular lumen (5). Secretion of MMPs by inflammatory cells and cardiomyocytes is associated with myocardial apoptosis (13, 38). Furthermore, the degradation of ECM components participates in inflammatory reaction by serving as a chemotactic trap for leukocytes (21, 29).

Experimental evidence has shown that MMPs are cytotoxic to several cell lines, such as neurons and fibroblasts, and increase cell death (32, 37). We demonstrate this phenomenon now in the cultured myocytes in the present study. Exogenous active MMP-1 caused myocyte injury in a dose- and time-dependent manner and increased cell death. Additionally, pretreatment of myocytes with a specific inhibitor of MMPs PD-166793 attenuated myocyte injury induced by MMP-1. However, the mechanism by which MMP-1 is cytotoxic remains undefined. It is possible that degradation of ECM, modulation of ion flux in cellular membrane (9), and activation of other cytotoxic cytokines, such as IL-1 (28) participate in the effects of MMP-1.

TGF-₁ and Ischemia-Reperfusion Injury and MMPs

It has been reported that TGF-₁ suppresses expression of MMP-9 and -12 in monocytes and macrophages in response to IL-1 and TNF- (11, 35). Because cardiac myocytes generate large amounts of MMP-1, and MMP inhibition has been shown to be cardioprotective (6, 8, 26, 27), we hypothesized that the cardioprotective effect of TGF-₁ may relate to the suppression of MMP-1. The present study indeed showed that a short period of ischemia-reperfusion was associated with a marked upregulation of MMP-1 mRNA and protein. Furthermore, treatment with exogenous TGF-₁ inhibited the upregulated expression of MMP-1 in ischemic-reperfused myocardium. In support of the role of MMPs in ischemic injury, target deletion and chemical inhibitors of MMPs have been shown to prevent infarcted myocardium from ventricular dilatation, aneurysm formation, and heart failure (8, 26, 27). Our observations suggest that inhibition of MMP-1 by TGF-₁ may be, at least in part, an important mechanism of cardioprotection by TGF-₁.

In conclusion, the present study suggests that TGF-₁ exerts cardioprotective effect during ischemia-reperfusion in the intact rat hearts. Part of the salutary effects of TGF-₁ may be related to inhibition of MMP-1 expression.

	ACKNOWLEDGEMENTS

This study was supported by a Merit Review grant from the Department of Veterans Affairs, a contract with the Department of Defense, funds from the Swedish Medical Research Council, and the Wrenette Worthen Williamson Cardiology Research Endowment.

	FOOTNOTES

Address for reprint requests and other correspondence: J. L. Mehta, Division of Cardiovascular Medicine, Univ. of Arkansas for Medical Sciences, 4301 West Markham St., #532, Little Rock, AR 72205-7199 (E-mail: MehtaJL{at}uams.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpheart.00992.2002

Received 18 November 2002; accepted in final form 19 December 2002.

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