Translating Koch’s Postulates to Identify Matrix Metalloproteinase Roles in Post-Myocardial Infarction Remodeling: The Cardiac Metalloproteinase Actions (CarMA) Postulates

Abstract

The first matrix metalloproteinase (MMP) was described in 1962; and since the 1990’s, cardiovascular research has focused on understanding how MMPs regulate many aspects of cardiovascular pathology from atherosclerosis formation to myocardial infarction and stroke. While much information has been gleaned by these past reports, to a large degree MMP cardiovascular biology remains observational, with few studies homing in on cause and effect relationships. Koch’s postulates were first developed in the 19^th century as a way to establish microorganism function and were modified in the 20^th century to include methods to establish molecular causality. In this review, we outline the concept for establishing a similar approach to determine causality in terms of MMP functions. We use left ventricular remodeling post-myocardial infarction as an example, but this approach will have broad applicability across both the cardiovascular and MMP fields.

Introduction

Heart failure that develops from myocardial infarction (MI) induced remodeling of the left ventricle (LV) is a major cause of morbidity and mortality, with approximately 70% of heart failure cases having MI as the etiology.^{1, 2} Occlusion of the coronary artery that leads to ischemia of sufficient duration to induce infarction is followed by a progressive physiological wound healing process that can evolve to prolonged pathological remodeling.³ The LV remodeling process incorporates the collective changes in size, shape, and function of the myocardium that follows the injury stimulus.³ LV remodeling is an intricate process that starts with an acute inflammatory response, overlapping with a proliferative phase, which in turn develops into a maturation phase.

In the absence of reperfusion, neutrophil influx initiates the inflammatory response and peaks by 24 h post-MI.³ At around day 3 post-MI, neutrophil infiltration is followed by the influx of macrophages. Macrophages stimulate fibroblast differentiation to myofibroblasts, triggering synthesis of large amounts of extracellular matrix (ECM) to generate the infarct scar.³ Infiltrating fibrocytes and monocytes can also transform into fibroblasts and myofibroblasts, contributing to post-MI remodeling.^4-6 Each phase of LV remodeling follows a closely orchestrated cascade to repair the myocardium. As a result, LV remodeling is a complex and intricate process. Co-morbidities and age are two factors that can interfere with the healing phase. Impaired LV remodeling is frequently caused by pathologic inflammation, which impairs deposition and maturation of ECM.⁷ The dynamic synthesis of ECM by cardiac fibroblasts and its breakdown by matrix metalloproteinases (MMPs) play essential roles in the successful remodeling of the LV post-MI.⁸

In addition to providing structural support for cells, the ECM plays a central role in the regulation of cellular functions.^9-11 ECM components include basic structural proteins such as collagen and elastin, and specialized proteins such as fibronectin, proteoglycans, and matricellular proteins. All ECM proteins can be degraded by one or more MMPs. Therefore, MMP levels and activity post-MI directly modulate ECM structure and composition, and consequently cardiac function.

The CarMA Postulates

In 1890, the German bacteriologist Robert Koch proposed three postulates to establish a causal relationship between a specific microbe and an infectious disease.^{12, 13} In this review, we have used Koch’s postulates as a framework to explain in parallel how we can depict the action of MMPs in LV remodeling (Figure 1). This schema will apply Koch’s postulates to the LV remodeling post-MI scenario as a specific example to illustrate the concepts, but this framework has broad implications to other cardiovascular pathologies as well as MMP functions in other systems. We propose the term cardiac metalloproteinase actions (CarMA) postulates to define this iterative process of proving MMP causality in LV remodeling. We will conclude our review with a discussion on how our postulates can help to drive the MMP field forward.

Figure 1.

A comparison of Koch’s postulates and the newly defined CarMa Postulates.

CarMA Postulate 1: MMP levels increase in all cases of MI in direct proportion to effect

The first Koch postulate requires that the microorganism be found in abundance in all organisms suffering from the disease, but should not be found in healthy organisms.^{12, 13} Our analogous postulate dictates that MMP protein expression likewise increases in all cases of MI, either in a linear relationship or over a threshold level of expression for cell types which have low MMP expression in the absence of MI. This postulate is observational, as it does not by itself show the direct effect; it merely places the suspect with the victim.

Human and animal models of MI have reported increased levels of several MMPs (Table 1). In particular, MMPs-1, -2, -3, -7, -8, -9, -13, and -14 levels increase; and even in the case of MMP-28, where total levels decrease post-MI, macrophage derived MMP-28 increases.^14-19 Most literature reporting MMP expression levels measure these MMPs in the infarcted region or in the plasma. MMPs -2, -8, -9, and-13 have been evaluated in both plasma and tissue.^20-22 It is important to note that 16 of the 25 MMPs identified to date have no literature regarding changes in levels post-MI, and absence of results means either that the results were negative or the studies have not been performed.

Table 1.

Matrix metalloproteinase (MMP) expression following myocardial infarction

MMP number	Other names	Level post- MI	Animal Model	Measured in humans	Method	References
MMP-1	Collagenase 1	LVI: ↑ Plasma: ↑	rat, mice	yes	immunoblot ELISA	28,132, 133 134
MMP-2	Gelatinase A	LVI: ↑ Plasma: ↑	rat, mice	yes	immunoblot, ELISA	28, 132, 133 135
MMP-3	Stromelysin 1	LVI: ↑	rabbit	no	zymography	41
MMP-7	Matrilysin	LVI: ↑	mice	no	immunoblot	14
MMP-8	Neutrophil collagenase; collagenase 2	LVI: ↑	rat sheep	no	immunoblot	45 46
MMP-9	Gelatinase B	LVI: ↑ Plasma: ↑	rat, mice, dog, rabbit,	yes	immunoblot, ELISA	28, 132, 133 47, 135-137
MMP-10	Stromelysin 2	Plasma: =		yes	ELISA	138
MMP-11	Stromelysin 3	Unknown
MMP-12	Macrophage elastase	Unknown
MMP-13	Collagenase 3	LVI: ↑	sheep	no	immunoblot	46
MMP-14	Membrane type (M↑)1- MMP	LVI: ↑	rat sheep	no	immunoblot	16 46
MMP-15	MT2-MMP	Unknown		no
MMP-16	MT3-MMP	Unknown		no
MMP-23	CA-MMP	Unknown		no
MMP-26	Matrilysin-2	Unknown		no
MMP-27		Unknown		no
Myocyte- derived MMP-28	Epilysin	LVI: ↓	mice	no	immunoblot	19
Macrophage -derived MMP-28		LVI: ↑	mice	no	immunoblot

LVI- left ventricle infarct region; CA-MMP – Cysteine Array Matrix Metalloproteinase

The increase in an MMP seen post-MI could be due to one of two reasons: a) there is an influx of cells not present in the normal myocardium that can express the MMP, or b) there is an upregulation of ectopic expression, such that cell types that normally do not express the specific MMP at high levels are now producing it. There is a lot of support for the first reason, with infiltrating neutrophils and macrophages being a predominant source for the upregulation of several MMPs post-MI, including MMP-8 and MMP-9. There is less support for the second reason, as cardiomyocytes, endothelial cells, and fibroblasts have not been carefully isolated and measurements made for per cell MMP concentrations. Recently, studies have focused on genetic defects leading to overexpression of activated MMPs and have identified MMP-related polymorphisms as a risk factor in the development of MI in humans. The following paragraphs summarize specific changes in individual MMPs in the post-MI setting, by clinical observations first followed by evidence in animal models.

Clinically, MMP-1 has mainly been studied in plasma. MMP-1 levels were higher in the plasma of patients with acute coronary syndrome.²³ MMP-1 serum levels predicted extent of coronary atherosclerosis and were significantly higher in male patients at 6 months compared to 4 days post-MI.^{24, 25} Interestingly, MMP-1 promoter polymorphisms -1607 1G/2G, -519 A/G, and -340 T/C have been associated with risk of early MI, although the authors did not examine what effect these polymorphisms had on MMP-1 levels.²⁶

MMP-1 is mainly expressed in leukocytes, fibroblasts, and endothelial cells in the post-MI LV. In rats, MMP-1 activity in the infarct LV begins at day 2, peaks at day 7, and declines through day 14 when activity levels return to baseline.^{27, 28} MMP-1 activity parallels the proliferative phase of tissue repair that occurs during myocardial healing. Fibroblasts and endothelial cells are the main cell types present during the proliferation phase, synthesizing new ECM proteins that will spatially replace dead myocytes and form de novo connective tissue to generate a vascularized infarct scar. The new blood vessels formed support the heavy cellular load and develop collateral circulation to the ischemic site. The formation of new vessels requires endothelial cell proliferation and degradation of multiple ECM proteins.²⁹ Studies on MMP-1 in the post-MI LV have been hampered by the fact that human MMP-1 is divergent from mouse MMP-1, in that the mouse has two MMP-1 isoforms: MMP-1a and MMP-1b.³⁰ In mice, MMP-1a shares 59% homology and MMP-1b shares 57% homology with human MMP-1.

MMP-2 is constitutively expressed under normal conditions and is synthesized by cardiomyocytes, endothelial cells, vascular smooth muscle cells, and fibroblasts.^31-33 Post-MI, MMP-2 levels increase both in human plasma and infarcted LV.³⁴ Myocytes and myofibroblasts are sources of MMP-2 post-MI.³⁵ Plasma MMP-2 levels were shown to strongly correlate with MI size and LV dysfunction in a ST-elevation MI population.³⁶ The MMP-2 1575 gene polymorphism, which increases MMP-2 levels in plasma, correlates with MI occurrence in a male Mexican population.³⁷

Peterson and colleagues reported MMP-2 mRNA and protein levels are elevated within 24 h post-MI and peak around day 14 post-MI in rats.¹⁶ In mice, MMP-2 activity rapidly increases within 4 days post-MI, peaks at day 7, and remains elevated until day 14.³⁸. Because MMP-2 has high constitutive activity, it has been thought of as the MMP housekeeping gene to oversee normal tissue turnover.³⁵

In a study of 271 patients under 45 years old, two MMP-3 related polymorphisms - Leu125Val PECAM1 and A1/A2 FVII - were identified as MI-related and showed strong influence in plaque formation.³⁹ A clinical study in adolescents with ventricular arrhythmia identified plasma MMP-3 as a biomarker of arrhythmia in patients with hypertrophic cardiomyopathy.⁴⁰

Animal models have shown MMP-3 levels to increase at day 2 post-MI in the myocytes of infarcted region and remain elevated through day 14 post-MI.^{35, 41} MMP-7 is also a biomarker for cardiac disease. Elevated serum MMP-7 levels were observed in 144 patients with LV hypertrophy, and MMP-7 was identified as a marker of LV structural remodeling.⁴²

Tissue analysis in animal model have shown elevated MMP-7 levels post-MI in the remote and infarcted myocardium.¹⁴ Interestingly, this MMP is expressed both in cardiomyocytes, which explains the increased levels in the remote tissue, and macrophages.¹⁴

MMP-8 is a major player during the inflammatory response. Studies in humans showed that increases in MMP-8 activity in the infarct area post-MI lead to increased susceptibility to cardiac rupture.⁴³ MMP-8 human plasma levels are significantly increased 1 day post-MI.⁴⁴

Early after injury, the major cellular source of MMP-8 is the neutrophil. As such MMP-8 expression levels in rats increase 6 fold after 6h and peak at 12h post-MI.⁴⁵ In sheep, MMP-8 has been shown to be expressed by macrophages during the later stages of remodeling.⁴⁶

In a clinical study of acute ST-segment elevation MI, plasma MMP-9 levels peaked on days 1 and 4 post-MI.⁴⁷. MMP-9 activity was positively correlated with LV volume.⁴⁸ Blankenberg and colleagues demonstrated that MMP-9 links to the development of LV dysfunction and late survival.^{20, 49, 50} This establishes that MMP-9 increases in direct proportion to the effect.

Of all the MMPs evaluated to date, MMP-9 has been the MMP most frequently tracked with the development of LV dysfunction. Rodent models have shown MMP-9 expression to increase post-MI, peaking at days 1-7 post-MI with neutrophils and macrophages being the main source of MMP-9 post-MI.^{16, 51}

Patients with pressure overload hypertrophy and a significantly reduced LV ejection fraction showed increased mRNA levels of MMP-1, -13, and -14.⁵² Other human studies have correlated increased levels of MMP-13 with cardiomyopathies.⁵³

Similarly to what is observed in humans, animal models have shown increased MMP-13 activity post-MI. MMP-13 is expressed in cardiac fibroblasts.²⁷ In a rat model, MMP-13 showed a biphasic profile, initially increasing 1-2 days post-MI followed by a second peak at 2 weeks.¹⁶ In an ovine model, MMP-13 levels were persistently increased up to 1 month post-MI.⁴⁶

The membrane-type MMP, MMP-14, slowly increases post-MI, peaking at 16 weeks in rats.¹⁶ In pigs, both expression and activity of MMP-14 increase post-MI.⁵⁴ MMP-14 is expressed in fibroblasts and myocytes.³⁵ In a sheep model of MI, MMP-14 increased in the border and infarcted regions compared with the control region and the levels significantly correlated with the extension of LV remodeling.⁴⁶ MMP-28 in normal mouse heart is mainly expressed in the cardiomyocyte, and as such total MMP-28 levels decrease early post-MI as a reflection of myocyte loss.¹⁹ While total levels decrease, the MMP-28 derived from macrophages increases from day 3 post-MI, when this cell type infiltrates into the infarct region.¹⁹

The results obtained to date highlight the fact that some MMPs increase (MMPs-1, -2, -3, -7, -8, -9, -13, -14 and macrophage derived -28) while others decrease (myocyte MMP-28). Of all that have been measured, all MMPs show changes from baseline values, which establishes the first postulate for these individual MMPs.

CarMa Postulate 2: MMP action can be mimicked in vitro

The second Koch postulate requires that the microorganism be isolated from a diseased organism and grown in pure culture.^{12, 13} In the case of MI, our postulate dictates that isolated cardiac cells stimulated with factors that induce MMPs display biological functions in vitro that are similar to what is observed during cardiac remodeling in vivo. The effect of MMP stimulation on isolated cardiac-related cells is summarized in Table 2. While the true translation of this postulate would be that treating cells with MMPs or MMP inhibitors would show the same effects as seen in vivo, studies in this arena have focused on stimuli that increase or decrease MMP levels and subsequently show an effect on cell function.

Table 2.

Effect of matrix metalloproteinases (MMPs) on isolated cells

Cells	Inducer/Inhibitors	Effect on MMPs	References
Cardiac Fibroblasts	Pro-Inflammatory Cytokines	↑ MMPs-1, -3, and -9	56
	TGF-β1	↑ MMP-2 and MMP-14	61
	Type I collagen lattice	↑ MMP-2, MMP-14, TIMP-2	66
	IGF-1	↑ MMPs 1, -2, -8, and -9	68
Human Cardiac Fibroblasts	TNF-α,	↑ MMP-1, -8, -3, -7 and -9	57, 64
Neutrophils	Post-ischemic cardiac lymph	↑ MMP-9	70
	PFA/LPS/PMA	MMP-8 translocation	72
	TNF-α and IL-8	↑ MMP-9	73-75
	Adenosine	↓ MMP-9	73-75
Human Neutrophils	TNF-α/LPS/PAF	↑ MMP -9	57, 64
Macrophages	PMA	↑ MMP-1 and MMP-3	78
	IFN-γ	↑ MMP-1 and MMP-3	78
Human Macrophages	IFN-γ/LPS	↑ MMP-1, -3, -7, -10, -12, -14 and -25, ↓ TIMP-3	77
	IL-4	↑ MMP -11, -12, -25 and TIMP-3, ↓ MMP-2, -8 and -19	77
	NF-κB	↑ MMP-1 and -3	79

IFN-γ – interferon-γ; IGF-1 – insulin-like growth factor-1; IL – interleukin; LPS - lipopolysaccharide; NF-κB - nuclear factor kappa B; PAF – platelet activating factor; PMA – phorbol myristate acetate; TGF-β₁ - transforming growth factor-β₁; TNF-α - tumor necrosis factor-α; TIMP - tissue inhibitor of metalloproteinase

Cardiac fibroblasts represent >50% of the cells in the normal mammalian heart, and fibroblast numbers dramatically increase post-MI.⁵⁵ Cardiac fibroblasts express MMPs-1, -2, -3, -7, -8, -9, and -14 under normal conditions.^{56, 57} MMP-2 followed by MMP-14 and MMP-1 were the most abundant MMPs in human cardiac fibroblasts.⁵⁷ MMPs -3, -7, -8, and -9 showed low expression.⁵⁷ MMP expression in fibroblasts isolated from post-MI LV remains to be evaluated.

Cardiac fibroblasts undergo a transition from a fibroblast to a myofibroblast phenotype during LV remodeling, and stimuli such as inflammatory cytokines that stimulate this phenotype transition also regulate the expression of several MMPs.^58-60 Transforming growth factor-β₁ (TGF-β₁) stimulates MMP-2 and MMP-14 expression in cardiac fibroblasts.⁶¹ MMPs -2, -9, and -14 can process TGF-β to its active form by processing latent TGF-β binding protein.^{62, 63} Treatment with tumor necrosis factor (TNF-α, a pro-inflammatory factor which plays a significant role in vivo in the genesis of post-ischemic inflammation, led to a pronounced increase in expression of MMP-3, -7, -8 and -9, while less than 2-fold increase was observed in MMP-1, -2, and -14.^{57, 64} In vitro, cardiac fibroblast survival is associated with increased expression and activity of MMP-2.⁶⁵ Cardiac fibroblasts cultured in a type I collagen lattice upregulate activated MMP-2 and MMP-14,.⁶⁶ Using a multitude of MMP null skin fibroblasts, the Weiss lab showed that secreted collagenases and gelatinases (MMPs -2, -8, -9, and -13) provide potent matrix-resorptive activity, while only MMP-14 was necessary for focal collagenolytic activity required for cell migration.⁶⁷ Insulin-like growth factor -1 (IGF-1), a factor released from its binding protein in the ECM by MMPs 1, -2, -8, and -9, modulates fibroblast function.⁶⁸ Treatment with IGF-1 increased fibroblast adhesion to several ECM proteins and induced the expression of collagen and integrins.⁶⁹ In summary, MMP expression and activity in cardiac fibroblasts can be induced in vitro resulting in modulation of cellular functions, such as adhesion, migration, cytokine production, and ECM secretion.

In addition to the cardiac fibroblast, the neutrophil and the macrophage are key inflammatory cell types that regulate LV remodeling post-MI. Isolated canine neutrophils incubated with post-ischemic cardiac lymph showed increased MMP-9 levels.⁷⁰ Neutrophil-derived MMP-8 activated lipopolysaccharide (LPS) induced CXC chemokines to regulate the initial inflammatory response and promote tissue responsiveness.⁷¹ Neutrophil activation in vitro leads to translocation of active MMP-8 from specific granules to the plasma membrane.⁷² Studies performed in neutrophils isolated from healthy donors showed that adenosine, a naturally produced nucleoside, inhibited release of MMP-9; whereas TNF-α and IL-8 stimulated MMP-9 expression.^73-75 Human neutrophils showed 10-fold increased expression of MMP-9 upon stimulation with pro-inflammatory mediators - TNF-α, LPS, or platelet activating factor.⁷⁶

Macrophages isolated from human blood and classically activated with interferon-γ (IFN-γ) and LPS showed up-regulation of MMPs-1, -3, -7, -9, -10, -12, -14 and -25.⁷⁷ Activation with interleukin-4 (IL-4) decreased MMP-2, -8, and -19 but increased MMPs-11, -12, and -25 steady-state mRNA levels.⁷⁷ Activation of monocytes/macrophages with phorbol myristate acetate (PMA) stimulated CD4+ T cells increased MMP-1 and MMP-3 expression which was inhibited by IFN-γ.⁷⁸ MMP-1, -3, and -9 increase with stimulation by factors that induce nuclear factor kappa B or activator protein-1 (ap-1).⁷⁹ Plasminogen regulates macrophage migration in inflammation by activation of MMP-9.⁸⁰

Based on these reports, several MMPs are elevated in isolated MI-relevant cells when stimulated with factors known to affect cardiac remodeling, which establishes the second postulate.

CarMA Postulate 3: Modulation of MMPs alter the course of cardiac remodeling

The third Koch postulate requires that the cultured microorganism cause disease when introduced into a healthy organism.^{12, 13} In the case of MI, our postulate decrees that interventions blocking or enhancing MMP functions will significantly affect LV remodeling.

Reviews by Lindsey et al. and Phatharajaree et al. discuss these studies extensively, and we have summarized those findings in Table 3.^{3, 48} A number of studies performed using broad spectrum MMP inhibitor have shown beneficial effects on cardiac remodeling post-MI.⁴⁸ The administration of the broad-spectrum MMP inhibitor, CP-471,474, in mice immediately after MI attenuated LV dilation at 4 day post-MI.⁸¹ This inhibitor inhibits MMP-1, -2, -3, -9, and -13.⁸¹ Another study in pigs showed that administration of an MMP inhibitor (PD166793) 5 days after MI led to reduction in MI size and expansion rate by 2 weeks post-MI.⁸² PD 166793 binds to the active domain of MMP-2, -3, and -13 with high affinity, and to MMP-1, -7, -9 and -14 with low affinity.⁸³ When treatment was continued for two months, the effect persisted to the late phase of MI healing, as evidenced by reduced LV chamber dilation.⁸²

Table 3.

Effect of matrix metalloproteinase (MMP) modulation on cardiac remodeling

Intervention	Model	Time post- MI effect observed	Effect	References
Broad spectrum MMP Inhibitor (CP-471,474)	mice	4 days	↓ LV dilation	81
Broad spectrum MMP Inhibitor (PD166793)	pig	5 days	↓ MI size and expansion	82
MMP Inhibitor (PGE- 530742)	pig	10 days	↓ LV end diastolic volume	53
Broad spectrum MMP Inhibitor (CP-471,474)	rabbit	4 weeks	↓ LV dilation, ↑ neovascularization	139
TISAM	mice	3 days	↑ survival, ↓ rupture	86
MMP-2 null	mice	28 days	↓ LV rupture	100
MMP-1 Tg	Mice	No additional injury	↑ systolic dysfunction, severe LV remodeling	102
MMP-2 Tg	mice	No additional injury	↑ systolic dysfunction, severe LV remodeling	103, 104
MMP-7 null	mice	7 days	↑ survival, preserved myocardial conduction patterns	111
MMP-9 null	mice	Up to 15 days	↓ LV enlargement, Collagen accumulation	101
Macrophage specific MMP-9 Tg	mice	5 days	↑ LV function, ↓inflammation	2, 105
MMP-28 null	mice	Up to 28 days	↑ LV dysfunction and rupture	15
MT1-MMP Tg	mice	Up to 14 days	↑ LV remodeling, fibrosis, ↓ survival	46
TIMP-1 null	mice	Up to 14 days	↑ LV remodeling	48
TIMP-2 null	mice	1 week	↑ Infarct expansion, LV dysfunction and inflammation	107
TIMP-3 null	mice	5 and 30 days	↑ LV rupture	140
TIMP-4 null	mice	3-7 days	↑LV rupture	109

I/R - ischemia/ reperfusion; LV- left ventricle; M – myocardial infarction; Tg- transgenic; TISAM –((2R)-2-[5-[4-[ethyl-methylamino]phenyl]thiophene-2-sulfonylamino]-3-methylbutyric acid

Yarbrough et al demonstrated that the MMP inhibitor PGE-530742, which blocks MMP-2, -3, -9 and -13, but not MMP-1 or -7, reduces progression of LV end-diastolic volume after MI.⁸⁴ PGE-530742 is a phosphoamide based inhibitor that binds with high affinity by forming a tetrahedral complex leading to MMP inhibition.⁸⁵ The relatively specific MMP-2 inhibitor, 2R-2-[5-[4-[ethyl-methylamino]phenyl] thiophene-2-sulfonylamino]-3-methylbutyric acid (TISAM), showed beneficial changes to remodeling. TISAM inhibits MMP-2, -9, and -14 but not MMP-1, -3, or -7. Inhibition of MMP-2 activity increased survival rate and reduced cardiac rupture and macrophage infiltration post-MI.⁸⁶ Tetracyclines such as doxycycline can also regulate coronary artery disease by inhibiting MMP -2, -8, -9, and -13 through their ability to chelate zinc.^87-89 Cardiac remodeling can be regulated by indirect modulation of MMP expression and activity. The cannabinoid receptor antagonist rimonabant decreased MMP-9 activity and TGF-β1 expression in rats, leading to reduced collagen content, attenuation of ECM destruction and fibrosis 6 weeks post-MI.⁹⁰ Salvianolic Acid A serves as a competitive inhibitor of MMP-9 and prevented LV remodeling post-MI, in part by preventing fibroblast proliferation and myofibroblast transdifferentiation.⁹¹ The mechanism by which MMP-9 modulates cardiac fibroblast proliferation and phenotype post-MI is not yet known.

Early administration of carvedilol in pigs with acute MI (AMI) showed reduced monocyte chemotactic protein-1 (MCP-1) and MMPs.⁹² Carvedilol is a beta blocker and vasodilator that has a unique property of guanine nucleotide modulatable binding.⁹³ Based on the mechanism of action of carvedilol, its effect on MMPs post-MI is probably indirect, but the exact molecular mechanisms that underlie these effects have not yet been established.⁹⁴ Inhibition of endothelin receptor type A by an ET_A-receptor antagonist prevented LV dilation in chronic post-MI rats through inhibition of MMP activation.⁹⁵ At 7 days post-MI, Wistar rats showed reduced collagen accumulation after treatment with an ET_A-receptor antagonist through an unknown mechanism.⁹⁶ TGF-β stimulates MMP-2 and -9 and inhibits MMP-1 and -3 synthesis in vitro, and in a rat model of ischemia-reperfusion TGFβ pre-treatment reduced LV dysfunction by blocking MMP-1 mediated cardiomyocyte necrosis.⁴⁸

MMP inhibition does have some negative effects, and clinical trials have highlighted the difficulties in separating out the divergent functions. MMP inhibition leads to delayed healing in vascular and dermal wounds, as MMP activity regulates the migration of inflammatory cells and smooth muscle cells into the wound.⁹⁷ MMP inhibition on collagen deposition has detrimental effects, as it prevents the accumulation of collagen necessary for scar formation.⁹⁷ MMP inhibition may affect expression or activity of other signaling factors, including TNF-α, TGF-β, and IL-1β, which can induce an imbalance in the ECM turnover process.⁹⁷ The timing of intervention to modulate MMP expression or activity plays a prominent role, as some MMP activities are essential for beneficial cardiac remodeling post-MI.⁴⁸

Several MMP null or transgenic mice have been generated to evaluate the effects of gene deletion and introduction in the post-MI setting.^{98, 99} Particularly, these studies are beneficial in understanding if global deletion of a specific MMP can rescue a disease phenotype, and if re-introducing the MMP at a determined time-point can alter disease development. MMP-2 null mice have attenuated LV rupture and reduced late remodeling post-MI.¹⁰⁰ MMP-7 null mice show improved survival post-MI through anti-arrhythmic effects mediated through connexin-43 preservation.¹⁴ MMP-9 null mice have attenuated LV enlargement and reduced collagen accumulation post-MI.¹⁰¹ MMP-28 deletion exacerbated LV dysfunction and rupture through a defective inflammatory response and suppressed M2 macrophage activation.¹⁵ More studies using genetic approaches are warranted to move the field towards clinical translation.

Transgenic MMP-1 or MMP-2 mice both develop a cardiac failure phenotype over time, even in the absence of superimposed injury.^102-104 Alternatively, transgenic overexpression of MMP-9 in macrophages attenuated the inflammatory response and improved LV function post-MI.² This report revealed that MMP responses in the MI setting are likely to be both beneficial and detrimental, depending on the time when the MMP is expressed and what substrates are nearby and available for processing. Cardiac restricted overexpression of MMP-14 caused adverse remodeling, increased fibrosis and reduced survival.^{2, 105}

In conjunction with this postulate, mechanisms that increase MMP activity should promote cardiac remodeling. The MMP inducer, EMMPRIN, increases in the LV of acute MI patients and may play a critical role in LV remodeling post-MI.¹⁰⁶ TIMP-1 deletion, as a mechanism to increase MMP activity, aggravated LV remodeling after MI, presumably through stimulating ECM turnover.⁴⁸ TIMP-2 null mice show greater infarct expansion, exacerbated LV dysfunction, and increased inflammatory response post-MI.¹⁰⁷ TIMP-3 and TIMP-4 deficiency leads to increased cardiac rupture.^{108, 109} MicroRNA miR-21 was recently identified to regulate MMP-2 and increase its expression in cardiac fibroblasts post-MI in an ischemia-reperfusion mice model via regulation of phosphatase and tensin homolog (PTEN) signaling pathway in infarct zone.¹¹⁰ Whether and how other miRNAs regulate MMPs has not been extensively explored.

Based on these reports, modulation of MMPs regulates LV remodeling, which establishes the third postulate.

CarMA Postulate 4: MMP proteolytic products regulate cardiac remodeling

The fourth Koch postulate requires that the microorganism be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.^{12, 13} In the case of MI, our postulate indicates that MMP substrate cleavage products should regulate LV remodeling, and that MMP effects can be rescued by altering substrate levels. For example, adding back substrate would rescue the MMP null phenotype, and removing substrate would rescue the MMP transgenic phenotype.¹¹¹ This postulate finalizes the complete mechanistic approach by explaining how MMPs function but is complicated by the fact that many MMPs increase post-MI and each MMP has multiple substrates, some of which remain to be identified.

The analogy to reisolation of a microorganism from a diseased individual would be to identify the MMPs associated with a particular cardiovascular disease. However, identification is not enough. The temporal profile of the MMP during the course of disease, as well as cellular source and substrates, are vital information for a full understanding of the disease and for development of diagnostic and prevention tools. Identification of MMPs and MMP-cleavage products as biomarkers in human patients may provide increased diagnostic capabilities for the early detection of disease. This is still a largely unexplored area in both the clinical and basic science fields of MMP research. However, the studies performed to date provide important information about MMP upregulation in patients.

MMP-3 and -9 and TIMP-4 concentrations in plasma samples of patients with atrial fibrillation (AF) showed predictive capabilities of these factors for the early recurrence of AF.¹¹² Stress induced cardiomyopathy (SIC) patients showed lower MMP-1 and -8 levels but higher TIMP-4 levels with elevated LV end-diastolic pressure compared to controls.¹¹³ In post-MI patients a specific plasma profile was observed, with decreased MMP-8 and -9 levels and increased TIMP-4 levels.¹¹⁴ The future identification of early changes in levels of MMPs in patients may help to reveal details about the functional mechanisms affected prior to the onset of cardiac disease.

Post-MI, ECM undergoes proteolysis directed by MMPs that leads to the generation of ECM peptide fragments called matricryptins or matrikines.¹¹⁵ Matricryptins are substrate fragments produced from the cleavage of such ECM proteins as collagens (I, IV, XVIII, and XV), connective tissue glycoproteins (fibronectin, thrombospondin-1, laminin, and secreted protein acidic and rich in cysteine (SPARC)), and elastin.¹¹⁶ Matricryptins serve as bioactive signaling molecules to regulate the post-MI inflammatory and scar formation responses.¹¹⁵

Most studies to date evaluating matricryptins roles have evaluated effects on angiogenesis, as a large number of angiogenic factors are released from ECM by MMPs. Endostatin, a fragment of collagen XVIII produced through proteolytic cleavage by elastase, suppresses adverse LV remodeling and heart failure in a rat MI model.¹¹⁷ Other angiogenic factors produced include angiostatin and tumstatin.

Fibronectin plays an important role in wound healing process post-MI and is an MMP substrate (MMP-7 and MMP-9, in particular).^{111, 118, 119} Fibronectin fragments influence remodeling by regulating monocyte migration into the infarcted myocardium which improves survival of injured cardiac myocytes.¹²⁰ Fibronectin fragments trigger the feedback mechanism to induce the fibronectin expression.¹¹⁹ A study in fibronectin extracellular domain (EDA) null mice showed that absence of the EDA domain improves survival and cardiac performance by regulating ECM turnover and inflammatory response post-MI.¹²¹

MMP substrates are not limited to ECM proteins. IL-1β is cleaved by MMP-9 to generate its active form.¹²² The proinflammatory cytokine IL-1β regulates LV remodeling post-MI.¹²³ Il-1β has also been shown to induce MMPs -1, -3, -8, -9, -13, and -14 in a number of cells and tissues, indicating a positive feedback mechanism.^{115, 123-125} IL-1β is associated with interstitial fibrosis in the chronic phase of a rat MI model.^123-125 Treatment with anti-IL-1β decreased expression of collagen type III in both infarct and non-infarcted remote areas.^123-125 In the acute phase of MI, anti-IL-1β treatment caused increased incidences of LV rupture and dilation.^123-125 IL-1β also coordinates with TNF-α to regulate ECM remodeling and fibrosis.¹²⁵

In addition to the substrates mentioned above, there are a wide variety of substrates that have been identified for MMP-9, including thrombospondins, laminins, tenascins, cytokines, and growth factors.⁵¹ However the effect of MMP-9 proteolysis of these substrates on LV remodeling has not been reported. Based on these reports, several cleavage products of MMPs regulate LV remodeling, which establishes the fourth postulate.

Challenging Dogma: Moving beyond Matrix Proteolysis

In the 21^st century, Fredricks and Relman suggested a revised version of Koch’s postulates based on the modern nucleic acid-based microbial detection methods.¹² The revised postulates provided more sensitivity and specificity in identifying the cause of the microbial disease. Similarly in the MMP field, further understanding is required to successfully predict how modifications could be beneficial or adverse to the post-MI response. The first postulate has been fairly well-defined, and we have a generally detailed understanding of the temporal and spatial patterns in the post-MI setting for about a third of the MMPs. The second, third, and fourth postulates still require large voids to be filled before we have a clear understanding of the mechanisms of MMP actions post-MI (Figure 2).

Figure 2.

Future Directions

There are several areas where additional in vivo and in vitro studies are needed, to provide a complete understanding of MMP mechanisms. For one, not all MMPs have been measured post-MI and their cell sources identified, which will tell us which cells produce which MMPs under which settings. Importantly, the interactions between a specific MMP and the particular cardiac cell involved remain to be examined. Previous studies have shown that although fibroblasts are the major source of MMPs, MMPs can also regulate fibroblast functions. MT1-MMP can cleave fibronectin and trigger fibrosis.¹²⁶ MMP-2 and MMP-9 cause collagen synthesis by regulating TGF-β.¹²⁶ Overexpression of TIMPs 1-4 also cause increased collagen synthesis and fibroblast differentiation.¹²⁶ These studies provide evidence of complex MMP and cardiac cell interactions and how they influence each other. Studies where MMPs themselves are used as input stimuli are needed, to compare with current studies where MMPs are measured as an output measurement. Cell based targeting studies are also needed that isolate cells directly from the post-MI LV to examine MMP levels under ex vivo conditions. These studies will explain how MMPs influence each other in different cell types and whether this interaction changes by cell type or time. This approach will also provide us information about which MMPs should be promoted or inhibited and at what times this intervention should occur. In order to precisely develop therapies to protect from adverse remodeling, more quantitative and mechanistic approaches are required to understand the dynamic interaction between MMPs and the different cell types.

MMP inhibition is one approach to delineate the role of a specific MMP in different pathological conditions. Catalytic MMP activity is regulated at four levels – gene expression, compartmentalization, zymogen activation, and enzyme inactivation.¹²⁷ Pathologically, MMPs are primarily regulated at the transcriptional level, with increased expression in response to hypoxia, cytokines, and growth factors. ^{33, 128} Inhibition of a specific MMP can be achieved with use of null mouse models, blocking peptides, and siRNAs. These methods will directly affect mRNA stability, protein translation, pro-MMP zymogen activation, trafficking, secretion and inhibitor binding.

While these approaches are promising, the failure of clinical trials to translate basic science findings has been frustrating. A more detailed understanding of MMP functions remains to be acquired. Apart from studying the effect of deletion and overexpression of MMPs in a post-MI heart, research should focus on understanding the effects of delivery of MMPs or MMP-specific inhibitors to the post-MI heart. A study in Fischer rats showed that injection of collagen to the infarcted heart improved LV stroke volume and ejection fraction.¹²⁹ Such localized treatment studies are required to delineate the processes of LV remodeling in the post-MI setting. Which MMPs target what ECM components at which days post-MI is still an unresolved question.¹³⁰ Since ECM turnover plays prominent role in affecting LV remodeling post-MI, techniques such as matridomic and degradomic proteomic approaches should be used extensively to sketch out the functions of each ECM component and to understand how these components are affected by the changes in MMP levels post-MI.¹³¹

A more complete understanding of MMP substrates is required, to know which substrates are cleaved in the post-MI setting, by which MMPs and at what time(s), and for what purpose. While there is a lot of information on some MMP substrates, such as those for MMP-2 and MMP-9, we do not know which substrates are the most relevant. For other MMPs, such as MMP-28, very few substrates have been identified to date. In addition, identifying MMP substrates is not enough. Knowledge on the biological functions of the MMP-generated substrate peptides is crucial. Identification of the MMP cleavage sites is required to understand the effect of MMP cleavage on the substrate and its potential for downstream signaling. Currently, only a few substrates and their cleavage sites are known. The MEROPS database integrates information of MMPs and their substrates, including known cleavage sites (merops.sanger.uk). One concept to be highlighted in this review is that MMPs process a wide range of substrates and thus affect myocardial biology in ways not only directly related to collagen structure. A more complete understanding of the MMP-substrate axis will likely identify specific substrates whose inhibition or overexpression could provide therapeutic means to prevent adverse LV remodeling. MMP substrates may also serve as useful diagnostic indicators to assess treatment response.

In conclusion, LV remodeling post-MI is a complex process regulated by a multitude of factors, including MMPs. Targeted studies are clearly warranted to identify the mechanisms of MMP actions at both in vitro and in vivo levels, which will help to improve outcomes for the post-MI patient. By suggesting a paradigm to establish cause and effect relationships between remodeling events and specific MMP actions, our postulates can help to further our understanding of both the MMP field generally and the MI field specifically.

Nonstandard Abbreviations and Acronyms

AF

Atrial Fibrillation

AMI

Acute Myocardial Infarction

AP-1

Activator Protein-1

CarMA

Cardiac Metalloproteinase Actions

CXC

CXC Chemokine

ECM

Extracellular Matrix

EDA

Extra Domain A

EMMPRIN

Extracellular Matrix Metalloproteinase Inducer

ET_A

Endothelin Receptor Type A

IFN-γ

Interferon-γ

IGF-1

Insulin-like Growth Factor-1

IL

Interleukin

I/R

Ischemia/Reperfusion

LPS

Lipopolysaccharide

LV

Left Ventricle

MCP-1

Monocyte Chemotactic Protein-1

MMP

Matrix Metalloproteinase

NF-κB

Nuclear Factor Kappa B

PAF

Platelet Activating Factor

PMA

Phorbol Myristate Acetate

PTEN

Phosphatase and Tensin Homolog

SIC

Stress Induced Cardiomyopathy

SPARC

Secreted Protein Acidic and Rich in Cysteine

Tg

Transgenic

TGFβ₁

Transforming Growth Factor-β₁

TNFα

Tumor Necrosis Factor-α

TIMP

Tissue Inhibitor of Metalloproteinase

TISAM

2R-2-[5-[4-[ethyl-methylamino]phenyl] thiophene-2-sulfonylamino]-3 methylbutyric acid