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. Author manuscript; available in PMC: 2014 Jan 1.
Published in final edited form as: J Thorac Cardiovasc Surg. 2012 Oct 25;145(1):267–277. doi: 10.1016/j.jtcvs.2012.09.071

Differential membrane type 1 matrix metalloproteinase substrate processing with ischemia–reperfusion: Relationship to interstitial microRNA dynamics and myocardial function

Shaina R Eckhouse a, Adam W Akerman a, Christina B Logdon a, J Marshall Oelsen a, Elizabeth C O’Quinn a, Elizabeth K Nadeau a, Robert E Stroud a, Rupak Mukherjee a, Jeffrey A Jones a, Francis G Spinale a,b
PMCID: PMC3602970  NIHMSID: NIHMS411608  PMID: 23102905

Abstract

Objectives

Membrane type 1 matrix metalloproteinase (MT1-MMP) is critical to a number of proteolytic and profibrotic events. However, upstream regulation of MT1-MMP with myocardial ischemia–reperfusion remains poorly understood. MicroRNAs regulate post-transcriptional events, and in silico mapping has identified a conserved sequence in MT1-MMP for microRNA-133a. This study tested the hypothesis that changes in microRNA-133a regulation occur with myocardial ischemia–reperfusion, which contributes to time- and region-dependent changes in MT1-MMP activity and processing of MT1-MMP substrates.

Methods

Yorkshire pigs (n = 12) underwent ischemia–reperfusion (90 minutes ischemia and 120 minutes reperfusion), where regional preload recruitable stroke work (sonomicrometry), interstitial MT1-MMP activity (microdialysis), Smad2 abundance (immunoblotting), and interstitial microRNA-133a (polymerase chain reaction) were determined within the ischemia–reperfusion and remote regions. Human left ventricular fibroblasts were transduced with microRNA-133a and anti–microRNA-133a (lentivirus) to determine the effects on MT1-MMP protein abundance.

Results

With ischemia–reperfusion, regional preload recruitable stroke work decreased from steady state (139 ± 20 mm Hg to 44 ± 11 mm Hg, P <.05) within the ischemia–reperfusion region. MT1-MMP activity increased in both regions. Phosphorylated Smad2 increased within the ischemia–reperfusion region. Both in vitro and in vivo interstitial levels of microRNA-133a decreased with ischemia and returned to steady-state levels with reperfusion. In vitro transduction of microRNA-133a in left ventricular fibroblasts decreased MT1-MMP levels.

Conclusions

Modulation of MT1-MMP activity and microRNA-133a exportation into the myocardial interstitium occurred in the setting of acute myocardial ischemia–reperfusion. In addition, changes in microRNA-133a expression in left ventricular fibroblasts resulted in an inverse modulation of MT1-MMP abundance. Therefore, targeting of microRNA-133a represents a potentially novel means for regulating the cascade of profibrotic events after ischemia–reperfusion.

Restoration of blood flow, or reperfusion, after myocardial ischemia can trigger a number of biological responses, including induction of the matrix metalloproteinases (MMPs), which can culminate in ischemia–reperfusion (I/R) injury and myocardial dysfunction.1-3 One particular MMP, membrane type 1 MMP (MT1-MMP), has been implicated in remodeling of the extracellular matrix (ECM) and is up-regulated in animal models of I/R.1-6 However, upstream regulation of MT1-MMP remains unknown. The microRNAs (miRs) play a role in regulating MT1-MMP expression by inhibiting translation or stimulating mRNA degradation.7 For example, changes in myocardial miR-29a levels were associated with ECM remodeling in the setting of I/R.8 Furthermore, changes in miR-133a levels were associated with modulation of ECM collagen.9,10 With the use of in silico mapping studies, a putative miR-133a binding site in the MT1-MMP transcript and in the transcripts of other members of the transforming growth factor-beta (TGF-β) pathway was identified.11-13 In addition, MT1-MMP may contribute to TGF-β activation through proteolysis of latent transforming growth factor binding protein 1 (LTBP-1).2,3,14 LTBP-1 proteolysis releases TGF-β, which promotes phosphorylation of Smads (Smad2) and downstream transcription of type I collagen.15,16 However, whether activation of TGF-β signaling cascade occurs with acute myocardial I/R injury has not been established. Therefore, this study tested the hypothesis that changes in miR-133a expression/abundance occur with acute myocardial I/R, coincident with time- and region-dependent changes in MT1-MMP substrate processing and activation of profibrotic pathways.

MATERIALS AND METHODS

Model of Ischemia–Reperfusion

Yorkshire pigs (n = 12, 30 to 35 kg; Hambone Farms, Orangeburg, SC) were instrumented for transient coronary artery occlusion.1,2 Briefly, a pair of sonomicrometry crystals and microdialysis probes were placed within the I/R region (posterior left ventricular [LV] wall) and in a defined remote region (anterior apical LV). Regional preload recruitable stroke work (rPRSW) was determined.1 After steady state, ischemia was induced for 90 minutes and then released for 120 minutes of reperfusion, and rPRSW was measured at 30-minute intervals. After the I/R protocol, the LV free wall was harvested for biochemical analysis. Pigs with no I/R (n = 5) were included as referent controls. Animals were treated and cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 1996). Additional information is provided in the Online Supplemental Methods.

Microdialysis and Fluorogenic Substrate Measurements

Microdialysis probes (20 kDa; CMA/Microdialysis, North Chelmsford, Mass) were placed in the mid-myocardium, and quenched fluorogenic peptides containing an artificial MT1-MMP-specific hydrolysis site or an endogenous MT1-MMP hydrolysis site from LTBP-1 were infused (3 μL/min) as previously described.1,3,17,18 Interstitial MT1-MMP activity or LTBP-1 hydrolysis was determined as a function of the fluorescence in the dialysate.

Immunoblotting

Immunoblotting was performed to determine myocardial levels of phosphorylated Smad2 (pSmad2) and Smad2 (antibodies 3101 and 5339, respectively; Cell Signaling, Danvers, Mass) and MT1-MMP (AB8221; Millipore, Billerica, Mass).1 The immunoreactive signals were analyzed using densitometric methods.

mRNA Quantification

Myocardial mRNA levels were determined using real-time polymerase chain reaction (PCR). Primer/probe sets were synthesized for porcine LTBP-1 and TGF-β receptor 1 (TGF-βR1), and purchased for MT1-MMP, MMP-9, type I collagen, and 18S rRNA (cat. no. Ss0339441_ml, Ss03392098_m1, Ss03373340_m1, and Hs99999901_s1, respectively; Applied Biosystems, Foster City, Calif).

Bioinformatics Analysis of microRNA-133a

In silico mapping of the 3′ untranslated region (UTR) of the human MT1-MMP gene (TargetScan Human 5.1, www.targetscan.org) identified a high-probability target for miR-133a (context score −0.17, PCt 0.34).11-13,19 Of note, the 8-mer seed sequence was perfectly replicated in the 3′-UTR of the porcine MT1-MMP gene. Moreover, when the human and porcine 3′-UTRs of MT1-MMP are aligned (ClustalW2; http://www.ebi.ac.uk/Tools/msa/clustalw2/), the miR-133a target nucleotide sequence matches with identity at 25 of 31 base pairs, suggesting that porcine MT1-MMPs also possess a high-fidelity target site for miR-133a. In similar fashion, miR-133a putatively targets several other genes that play an important role in the profibrotic response, including MMP-9, LTBP-1, TGF-βR1, and type I collagen.9,10,13

microRNA Quantification

miR levels were determined in myocardial homogenates, microdialysate samples, and conditioned media samples (100 μL) from the in vitro studies. Isolated RNA was reverse transcribed, and expression levels of miR-133a and miR-29a were determined.

Simulated Ischemia–Reperfusion in Porcine Left Ventricular Fibroblasts

Porcine LV fibroblasts were incubated for 120 minutes in normoxic medium (O2 tension >250 Torr).20 The dishes were then incubated in hypoxic medium (2 mL), which was infused with 95% nitrogen, 5% CO2 for 30 minutes (O2 tension <50 Torr). Next, the cells were reincubated in normoxic medium for 120 minutes. Conditioned medium was collected at the end of each defined period and assayed by real-time polymerase chain reaction for exported miRs.21

In Vitro Membrane Type 1 Matrix Metalloproteinase Protein Modulation

Human LV myocardial fibroblasts (2 × 105 cells, n = 3) were transduced with 1 of 2 lentiviral vectors to overexpress miR-133a (pMIRNA1-hsa-miR-133a-1) and for knockdown of miR-133a (pmiR-Zip-hsa-miR-133a-1; both System Biosciences, Mountain View, Calif). Five days after transduction, the fibroblasts were collected for determination of MT1-MMP protein levels.

Data Analysis

LV regional function and hemodynamics were compared between groups by analysis of variance (ANOVA). Interstitial MT1-MMP activity and LTBP-1 substrate hydrolysis were expressed as a percent change from steady-state values (steady-state set to 0). The integrated optical density values for pSmad2, Smad2, and MT1-MMP were expressed as a percent of controls and compared using a 1-sample t test. Levels of miRs and mRNA expression levels were normalized to 18S or Caenorhabditis elegans miR-39 values, as appropriate, and compared as a fold change from control values by the ΔΔCt method.22 Post hoc mean separation after each ANOVA was performed using Bonferroni-adjusted pairwise comparisons. Statistical analyses were performed using STATA statistical software (StataCorp, College Station, Tex). Results are presented as the mean ± standard error of the mean.

RESULTS

LV function and hemodynamics at steady state and with I/R are summarized in Table E1. Although heart rate increased during reperfusion,1,2 LV peak systolic and mean arterial pressures remained unchanged with I/R. This suggests that the I/R protocol did not cause significant hemodynamic compromise. However, regional segmental shortening and rPRSW decreased in the I/R region with ischemia and remained decreased during reperfusion (Table E1 and Figure 1, A, respectively). Interstitial MT1-MMP activity increased over time in both the I/R and remote regions (Figure 1, B). However, interstitial MT1-MMP–mediated LTBP-1 substrate hydrolysis increased only within the I/R region (Figure 1, C).

FIGURE 1.

FIGURE 1

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A, Regional myocardial contractile function. Time-dependent rPRSW was determined in the remote (solid dot) and I/R (open triangle) regions. A time- and region-dependent difference was observed (ANOVA; F = 3.82 and F = 27.74, respectively; P <.01). B, MT1-MMP–specific fluorogenic peptide hydrolysis. A time- and region-dependent difference was observed (ANOVA; F = 9.50 and F = 3.33, respectively; P < .05). C, LTBP-1 substrate hydrolysis. A region-dependent difference was observed (ANOVA, F = 10.20, P <.05). *P < .05 versus steady state. I/R, Ischemia–reperfusion; rPRSW, regional preload recruitable stroke work; MT1-MMP, membrane type 1 matrix metal-loproteinase; LTBP-1, latent transforming growth factor binding protein 1.

Total Smad2 abundance increased in the I/R region after I/R (Figure 2, A). A small increase in pSMAD2 was measured in the remote region, whereas a robust increase was observed in the I/R region after I/R (P<.05 vs referent control) (Figure 2, A).

FIGURE 2.

FIGURE 2

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Activation of TGF-β signaling pathway. A, Immunoblotting total Smad2 and pSmad2 in the remote region, I/R region, and controls (n = 5). *P<.05 versus control. ‡P<.05 versus remote region. B, Myocardial mRNA levels for MMP9, MT1-MMP, LTBP1, TGF-βR1, and COL1A1 (type I collagen) in the remote region, I/R region, and controls. A region-dependent difference was observed (ANOVA; F = 4.45, F = 14.37, F = 14.62, F = 9.69, F = 6.48, respectively; all P<.05). *P<.05 versus control. IR, Ischemia–reperfusion; LTBP1, latent transforming growth factor binding protein 1; MMP9, matrix metalloproteinase 9; MT1-MMP, membrane type 1 matrix metalloproteinase; pSMAD2, phosphorylated Smad2; TGF-βR1, transforming growth factor beta receptor 1.

Myocardial mRNA levels of essential ECM-modifying genes after I/R injury are shown in Figure 2, B. Relative mRNA levels of MT1-MMP, MMP-9, LTBP-1, TGF-βR1, and COL1A1 were increased within the I/R region, whereas LTBP-1, TGF-βR1, and MT1-MMP mRNA levels were increased in the remote region.

With ischemia, interstitial miR-133a levels decreased in the both the I/R region and the remote region (Figure 3, A, top and bottom). With reperfusion, interstitial miR-133a levels returned to steady state within the I/R region and remained decreased in the remote region. Interstitial miR-29a levels remained unchanged in either region with I/R (Figure 3, B, top and bottom). At the completion of I/R, total myocardial miR-133a and miR-29a levels were similar in the remote region, I/R region, and referent controls (ΔΔCt; miR-133a 0.99 ± 0.67 and 1.22 ± 0.96, respectively, P =.50; ΔΔCt; miR-29a 1.12 ± 0.51 and 1.18 ± 0.62, respectively, P =.78). With simulated I/R, levels of miR-133a in the conditioned medium decreased with hypoxia (P =.047) and returned to steady state after reperfusion (Figure 4, A).

FIGURE 3.

FIGURE 3

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Interstitial miR levels. Interstitial levels of miR-133a and miR-29a were measured at steady state (gray), ischemia, and after reperfusion for the I/R (white) and remote (black) regions. Top: absolute values. Bottom: Data are depicted as a percent change from steady state. A, Interstitial miR-133a. A time-dependent difference was observed (steady-state miR-133a = 3.23 × 10−3 ± 1.15 × 10−3; ANOVA; F = 3.50; P<.05). B, Interstitial miR-29a. No change with I/R was observed (ANOVA, F = 0.39, P<.68). *P<.05 versus steady state. ‡P<.05 versus ischemia. miR, MicroRNA.

FIGURE 4.

FIGURE 4

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In vitro studies. A, Levels of miR-133a in conditioned medium from porcine LV fibroblast cultures (n = 3) under normoxia, hypoxia, and reoxygenation as a function of normoxia levels. B, MT1-MMP protein abundance in normal human LV fibroblast cultures (n = 3) 5 days after transduction with miR-133a or anti-miR-133a as a percent change from referent control values. *P<.05 versus steady state. ‡P<.05 versus ischemia. miR, MicroRNA; MT1-MMP, membrane type 1 matrix metalloproteinase.

In normal human LV fibroblasts, overexpression of miR-133a caused a reduction in MT1-MMP protein levels (Figure 4, B). Conversely, knockdown of miR-133a by anti–miR-133a caused an increase in MT1-MMP protein abundance.

DISCUSSION

Myocardial I/R can elicit a number of cellular and extracellular events, including contractile dysfunction, localized release of bioactive molecules, and induction of proteolytic pathways, which includes changes in MT1-MMP abundance and activity.1,2 The miRs are a major determinant of post-transcriptional regulation; certain miRs, such as miR-133a and miR-29a, have been identified as important mediators in ECM remodeling in the setting of I/R.8-10 Accordingly, this study tested the hypothesis that changes in miR-133a regulation occur with acute myocardial I/R injury, which contributes to time- and region-dependent changes in MT1-MMP activity processing of MT1-MMP substrates. The significant findings of the present study are as follows: (1) The contractile deficit that occurred with I/R was associated with increased MT1-MMP activity in both the remote and I/R regions and increased hydrolysis of the MT1-MMP substrate, LTBP-1, in only the I/R region. Concomitantly, there were differential and dynamic changes in interstitial levels of miR-133a without a change in the total myocardial pool of miR-133a during I/R. (2) Simulated I/R in myocardial fibroblasts recapitulated the in vivo changes in miR-133a levels, suggesting a direct role for I/R in the exportation of miR-133a. (3) miR-133a transduction in myocardial fibroblasts resulted in a decrease in MT1-MMP protein levels. (4) There was an acute activation of the TGF-β signaling pathway, components of which include many targets of miR-133a. Therefore, these findings suggest that changes in miR-133a levels during acute I/R may potentiate long-term sequelae of TGF-β signaling with respect to myocardial fibrosis and remodeling. Thus, modulation of miR-133a represents a potentially novel molecular target for regulating a cascade of TGF-β–mediated profibrotic events after I/R.

Coronary revascularization after acute coronary syndrome is accompanied by I/R injury, which can result in regional contractile dysfunction that gives rise to altered stress/strain patterns during the cardiac cycle, as well as the acute elaboration of a number of bioactive molecules.2,23 These multifactorial changes lead to LV remodeling, characterized by LV dilation and alterations in the content and composition of the myocardial ECM.1,23,24 In the present study, regional contractile dysfunction occurred with I/R within the I/R region and transiently in the remote region. The finding of persistent contractile dysfunction despite reperfusion within the I/R region and activation of a key proteolytic pathway mediated by MT1-MMP is consistent with past studies.1,2,23 The present study builds on these past reports by demonstrating that key upstream regulators and downstream effectors of altered MT1-MMP activity are induced acutely with I/R. Specifically, processing of proteins that contribute to fibrosis (LTBP-1 and TGF-β) and changes in interstitial miR content was altered in the setting of I/R. However, it must be recognized that alterations in loading conditions also may have served as a stimulus for increased MT1-MMP activity and LTBP-1 substrate hydrolysis at the I/R region. First, changes in mechanical loading can alter MT1-MMP induction.25-28 For instance, mechanical stretch of murine aortic rings increased activation of the MT1-MMP gene promoter.27 Likewise, increased mechanical loading of papillary muscles increased MT1-MMP expression.28 Second, changes in mechanical loading can change the conformation of the cell surface integrins, which in turn may induce a conformational change in LTBP-1 through its integrin binding site and release of TGF-β.29 The source of acute changes in interstitial MT1-MMP activity notwithstanding, the results from the present study demonstrate that long-term sequelae of I/R, with respect to changes in contractile function and LV remodeling, are likely initiated by these molecular events in the early I/R period.

The miRs, which are post-transcriptional regulatory molecules, contribute to modulation of ECM remodeling through regulation of processes, which in turn, contribute to fibrosis and ECM degradation.8-10 For example, overexpression of miR-133a decreased collagen production in cardiac fibroblasts.9 In mice with cardiac-restricted miR-133a overexpression, myocardial fibrosis was attenuated in the setting of pressure overload hypertrophy.10 Also, miR-29a knockdown was associated with increased mRNA expression of fibrillar collagen in mice and a reduction of infarct size in rats.8,30 An association between miR-133a and several MMPs has been described in the setting of hematologic and cardiovascular diseases.30-35 For example, a down-regulation of miR-133a was associated with increased plasma levels of several MMP types in patients.35 With the development of ischemic heart disease, past studies have reported a decrease in miR-133a and an increase in MT1-MMP protein abundance.1,31,34 Taken together, the findings from these past studies and results from mapping of a binding site for miR-133a on the MT1-MMP transcript formed the rationale for examining the post-transcriptional regulation of MT1-MMP by miR-133a in the context of myocardial I/R injury. The present study builds on these past reports by demonstrating that myocardial fibroblasts synthesize and release miR-133a and that changes in miR-133a levels can modulate MT1-MMP protein abundance. Specifically, MT1-MMP abundance was reduced after miR-133a overexpression in LV fibroblasts, and conversely, MT1-MMP abundance increased after LV fibroblasts were transduced with anti-miR-133a. Together, these findings suggest a role for miR-133a–mediated regulation of MT1-MMP abundance (Figure 5) in the context of myocardial I/R.

FIGURE 5.

FIGURE 5

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Working hypothesis regarding MT1-MMP activation and changes in interstitial miR-133a with I/R. A, At steady state, MT1-MMP mRNA is translated and ultimately transported to the membrane, yielding a competent transmembrane protease. The present study identified a measurable amount of miR-133a exportation into the myocardial interstitial space, and MT1-MMP protein abundance can be regulated by miR-133a. B, With acute ischemia, interstitial MT1-MMP activity increases, likely because of increased trafficking to the membrane.1 Interstitial miR-133a levels decreased, suggesting decreased exportation and increased intracellular concentrations, where post-transcriptional regulation of its targets can occur. C, With subsequent reperfusion, interstitial MT1-MMP activity remained increased while interstitial miR-133a returned to steady-state levels within the I/R region, but exportation was not restored in the remote region. Therefore, increased miR-133a exportation within the I/R region during reperfusion (ie, a decrease in the intracellular pool of miR-133a) would relieve translational repression of miR-133a targets, including those involved with the TGF-β signaling pathway. miR, MicroRNA; MT1-MMP, membrane type 1 matrix metalloproteinase.

Past in vitro studies have reported that miRs may be imported/exported from cells,21 and a recent study reported changes in total miR-133a levels in a rodent model of I/R.36 Building on these past findings, the present study demonstrated that a quantifiable pool of miR-133a and miR-29a was present in the myocardial interstitium. In addition, interstitial miR-133a levels changed while the total myocardial pool of miR-133a remained unaltered, suggesting that miR exportation is regulated with I/R (Figure 5). Of note, interstitial levels of miR-29a remained unchanged in the setting of I/R, suggesting that miR-133a export is selective and I/R specific. To examine this further, primary porcine LV fibroblasts were exposed to a simulated I/R protocol, and miR-133a within the conditioned media decreased with hypoxia and returned to steady-state levels with reoxygenation, in concordance with the in vivo microdialysis findings. Therefore, the present study provides evidence that differential and dynamic changes in extracellular miR content occur in the setting of acute myocardial I/R.

It is important to note that miRs or closely related miR families can target multiple mRNA transcripts involved in common biological signaling cascades.37 For example, in silico mapping studies of miR-133a have demonstrated several putative targets involved in both ECM degradation, such as MMP-9 and MT1-MMP, and TGF-β–mediated fibrosis, such as MT1-MMP, LTBP-1, COL1A1, and TGF-βR1. In animal and clinical studies, an abundance of several MMPs is generally increased with myocardial ischemia, specifically MMP-9 and MT1-MMP.1,2,38 Although changes in levels of both MMP-9 and MT1-MMP are generally associated with inflammatory cell infiltration, both can be produced by endogenous cells resident in the myocardium and are involved in a number of proteolytic events within the ECM.1-4,6,14,24 For example, MT1-MMP is a membrane-bound MMP fundamental in activating MMP-2 at the cell surface,5 and previous large animal studies have measured increased MT1-MMP with I/R injury.1,2 MT1-MMP also is involved in pericellular proteolysis and the release of ECM growth factors from sequestration sites.4 In the present study, interstitial MT1-MMP activity was increased in both the I/R and the remote regions. In a past study that examined the effects of an antecedent MI that was followed by acute I/R injury at the site remote from the original MI, interstitial MT1-MMP activity within the remote myocardial region remained unaltered for the duration of the I/R period.2 In contrast, a small, but significant, increase in MT1-MMP activity within the remote region during the I/R period was observed in the present study. However, it must be recognized that there are at least 2 differences with respect to the experimental design between these 2 studies: First, the coronary artery bed perfusing the “remote” myocardial region was different between the 2 studies. Specifically, the remote region was perfused by the left anterior descending coronary artery in the present study and through the circumflex arterial system in the past study.2 A second, and perhaps more important, difference was the time that ischemia was maintained. In the past study, ischemia was maintained for 60 minutes as opposed to the 90-minute ischemic period in the present study.2 Although this remains speculative, it is possible that the increase in ischemic time may have been sufficient to manifest in detectable changes in MT1-MMP activity within the remote myocardial region. Indeed, long-term ischemia (ie, myocardial infarction, as demonstrated in the same past study) was associated with increased MT1-MMP activity in the region remote from the MI.2 Therefore, the difference with respect to induction of MT1-MMP activity within the remote region between the 2 studies may have been due to regional differences, as well as the time for which ischemia was maintained. Nevertheless, the present study demonstrated that after I/R, mRNA levels of MMP-9 and MT1-MMP also were increased, suggesting that acute induction of pathways contributing to ECM proteolysis accompanies I/R. In vitro studies have provided evidence for MT1-MMP–mediated proteolysis of LTBP-1, resulting in the release of TGF and increased activation of profibrotic pathways.3,14 In mice with cardiac-restricted overexpression of MT1-MMP, enhanced proteolytic processing of LTBP-1 was reported and associated with elevated TGF-β–mediated Smad2 activation and increased myocardial fibrosis.3 A novel finding of the present study was that MT1-MMP–mediated LTBP-1 substrate proteolysis increased only in the I/R region. This region-dependent MT1-MMP–mediated LTBP-1 substrate proteolysis could set the stage for increased TGF-β signaling within the I/R region. Consequently, protein and mRNA levels of components in the TGF-β signaling pathway were increased within the I/R region. Specifically, the abundance and phosphorylation of Smad2 (pSmad2) were increased in the I/R region concomitant with increased mRNA expression of LTBP-1, TGF-βR1, and COL1A1. Therefore, these results suggest that an acute, region-dependent activation of TGF-β signaling and transcriptional induction of profibrotic molecules occurred with I/R and may promote fibrosis through the subsequent production of ECM proteins. For example, in a pig model of myocardial I/R, Barallobre-Barreiro and colleagues39 reported an increased abundance of fibrillar collagen within the I/R region at 15 and 60 days after the initial ischemic injury. Moreover, previous studies have demonstrated that miR-133a can play a regulatory role in the determination of collagen content and connective tissue growth factor abundance, 2 components that are important in myocardial fibrosis.9,10 The present study demonstrated that interstitial signaling in response to I/R activates both proteolytic and profibrotic pathways, which are initiated at the time of the acute I/R event and can potentiate LV remodeling over time. Specifically, interstitial miR-133a decreased within both the remote and I/R regions with ischemia, suggesting maintenance/increase of intracellular miR-133a, which could attenuate translation of MT1-MMP and the components of the TGF-β signaling pathway.9,10 With reperfusion, extracellular miR-133a increased from ischemia to reperfusion, suggesting a restoration of miR-133a export (or decrease in intracellular miR-133a) in the I/R region, but not in the remote region (Figure 5). These findings suggest a potential loss of miR-133a–mediated regulation of remodeling processes within the I/R region. Therefore, it is likely that the transient changes in miR expression during the I/R period, as observed in the present study, set into motion a sequence of events that trigger changes in the abundance/activity of proteins and activation of signaling cascades that result in a fibrotic response. However, the present study was focused on determining whether changes in miR abundance could be detected within the myocardial interstitium during the acute phase of I/R, and not on long-term evaluation of interstitial miR-133a and its downstream targets. Therefore, future studies would be required to determine the effect of the changes in miR-133a levels that occurred with acute I/R on the abundance of myocardial proteins in the ischemic and remote myocardial regions when studied over longer (days/months) reperfusion periods.

The present study is not without limitations. It must be recognized that miR-133a may bind to gene sequences for a number of proteins, including those for MMP-9, MT1-MMP, LTBP-1, TGF-βR1, TGF-β, and connective tissue growth factor.11-13 Conversely, translation of these proteins also may be regulated by a number of other miRs.37 A number of cell types within the myocardium, including myocytes and fibroblasts, are likely sources of miR-133a.9,10,36,40 The rationale for examining the role of myocardial fibroblasts with respect to changes in MT1-MMP and miR-133a with I/R was 2-fold: First, fibroblasts are the most numerous cell type within the myocardium and contribute to myocardial remodeling through the synthesis and release of MMPs, tissue inhibitors of matrix metalloproteases, and components of the ECM.41,42 Second, the region of a myocardial infarct is often densely populated with fibroblasts because of loss of myocytes secondary to ischemic injury.43 The in vitro studies using simulated hypoxia-reoxygenation in isolated myocardial fibroblasts recapitulated the in vivo findings of I/R with respect to changes in miR-133a levels. This finding suggests that myocardial fibroblasts contributed, at least in part, to changes in interstitial levels of miR-133a with I/R. Finally, on the basis of the finding that no differences in the total pool of miR levels were noted among the remote region, I/R region, and control myocardium, changes in interstitial miR-133a levels likely reflected changes in intracellular miR-133a levels. However, whether changes in interstitial miR levels occurred because of changes in viability of cells in the I/R region was not determined.

CONCLUSIONS

The findings of the present study demonstrated I/R-dependent changes in MT1-MMP activity and TGF-β signaling. These changes coincided with dynamic alterations in miR-133a exportation into the myocardial interstitium and may identify miR-133a as a novel target for modulating the long-term sequelae that develop after a clinically relevant I/R event.

Supplementary Material

Acknowledgments

This work was supported by National Institute of Health Grants HL057952, HL059165, and HL095608, and a Merit Award from the Veterans’ Affairs Health Administration. S.R.E. was supported by National Institute of Health Grant T32 HL007260.

Abbreviations and Acronyms

ANOVA

analysis of variance

ECM

extracellular matrix

FGM

fibroblast growth medium

I/R

ischemia–reperfusion

LTBP-1

latent transforming growth factor binding protein 1

LV

left ventricular

miR

microRNA

MMP

matrix metalloproteinase

MT1-MMP

membrane type 1 matrix metalloproteinase

PCR

polymerase chain reaction

pSmad2

phosphorylated Smad2

rPRSW

regional preload recruitable stroke work

TGF-β

transforming growth factor-beta

TGF-βR1

transforming growth factor beta receptor 1

UTR

untranslated region

Footnotes

Supplemental material is available online.

Disclosures: Authors have nothing to disclose with regard to commercial support.

Read at the 92nd Annual Meeting of The American Association for Thoracic Surgery, San Francisco, California, April 28-May 2, 2012.

References

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