Prognostic impact of matrix metalloproteinase gene polymorphisms in patients with heart failure according to the aetiology of left ventricular systolic dysfunction
Frédérique Mizon-Gérarda,
Pascal de Grooteb,
Nicolas Lamblina,b,
Xavier Hermanta,
Jean Dallongevillea,
Philippe Amouyela,
Christophe Bautersa,b,* and
Nicole Helbecquea
a INSERM U508, Institut Pasteur de Lille, 1 rue Calmette, 59019 Lille cedex, France
b Hôpital Cardiologique, Service de Cardiologie C, Centre Hospitalier Universitaire de Lille, Place de Verdun, 59037 Lille cedex, France
Received April 10, 2003;
revised January 14, 2004;
accepted January 22, 2004
* Corresponding author. Tel.: +33-3-20-44-50-45; fax: +33-3-20-44-48-81
E-mail address: cbauters{at}chru-lille.fr
See page 631 for the editorial comment on this article1
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Abstract
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Aims To assess the possible effect of functional polymorphisms in matrix metalloproteinase (MMP) gene promoters on the clinical outcome of patients with heart failure.
Methods and results We studied 444 consecutive patients who were referred to our centre for evaluation of left ventricular dysfunction. We extracted genomic DNA from white blood cells and determined the 1306 C
T MMP-2, 1171 5A
6A MMP-3, and 1562 C
T MMP-9 polymorphisms. Clinical follow-up (median 717 days) was obtained for 443 patients. The MMP-3 polymorphism had a different impact on cardiac survival in HF patients with ischaemic and non-ischaemic cardiomyopathy (interaction
). The MMP-3 5A/5A genotype was an independent predictor of cardiac mortality (HR 2.92 [1.236.69];
) in patients with non-ischaemic HF. In contrast, there was no evidence for any effect of the MMP-3 genotype on cardiac events in patients with ischaemic cardiomyopathy. The MMP-9 polymorphism was associated with cardiac survival
independently of HF aetiology. In multivariate analysis, the MMP-9 T allele was an independent predictor of cardiac mortality (HR 1.81 [1.093.02];
). Finally, there was no evidence for any association between MMP-2 polymorphism and cardiac survival.
Conclusion MMP-3 and MMP-9 polymorphisms contribute to variability in cardiac survival in HF patients. These data suggest that MMP genotyping could provide important additional information for refining risk stratification in patients with heart failure. MMP genotyping may help to select patients who could benefit from MMP inhibition.
Key Words: Heart failure Polymorphism Genetic Survival Matrix Remodelling
List of Abbreviations: ACE: angiotensin-converting enzyme ECM: extracellular matrix HF: heart failure LV: left ventricular LVEDD: left ventricular end-diastolic diameter LVEF: left ventricular ejection fraction MMP: matrix metalloproteinase NYHA: New York Heart Association VO2: oxygen consumption
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Introduction
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Left ventricular (LV) remodelling is a key determinant of long-term survival in heart failure (HF). Changes in the myocardial extracellular matrix (ECM) play an important role in the remodelling process.1 Among the pathways that contribute to ECM remodelling, the activation of a family of proteolytic enzymes known as the metalloproteinases (MMP) appears to be particularly important.25 This proteolytic system degrades a wide spectrum of ECM proteins and is constitutively expressed in a large number of cell and tissue types, including myocardium.
In animal models, an increase in myocardial MMP levels is associated with progression of LV dilation and dysfunction.3,4 In men, several studies have demonstrated increased MMP activity in myocardial samples from patients with congestive HF.2,5 Selective induction of MMP species occurs within the failing human myocardium. Moreover, the concentration of MMP in human failing heart depends on the aetiology of HF. MMP-9 (gelatinase B) myocardial levels are elevated in both ischaemic and non-ischaemic HF, whereas MMP-2 (gelatinase A) and MMP-3 (stromelysin 1) myocardial levels are high in non-ischaemic but not in ischaemic HF.5
In the present study, we hypothesised that a genetically mediated increase in proteolysis in the myocardium may worsen clinical outcome in HF patients. Moreover, since MMP induction depends on the underlying HF aetiology, we anticipated that this effect may be different in patients with ischaemic and non-ischaemic HF. We studied three polymorphisms in the promoters of the MMP-2, MMP-3, and MMP-9 genes; these polymorphisms affect MMP expression in vitro 68 and in vivo,9,10 and may consequently play a role in the regulation of extracellular matrix proteolysis. We thus designed the present study to test the hypothesis that these genetic variants may alter the outcome of patients with ischaemic or non-ischaemic HF.
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Methods
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Study population
We included 444 unrelated consecutive patients who were referred to our centre between January 1998 and December 2001 for evaluation of LV systolic dysfunction. Patients were included if they were ambulatory, stable for at least 2 months, and had a left ventricular ejection fraction (LVEF)
45%. Patients were excluded if they had a recent (
3 months) myocardial infarction, unstable angina, or coronary revascularisation. As part of the prognostic evaluation, patients underwent echocardiography, radionuclide angiography, and cardiopulmonary exercise testing as previously described.11 In addition, patients underwent systematic cardiac catheterisation to help define the aetiology of LV dysfunction. Patients were classified as having an ischaemic aetiology if they had experienced a previous myocardial infarction and/or had significant (
50%) coronary artery disease at angiography. Nine patients refused coronary angiography and were classified as having an unknown aetiology.
At the time of entry into the study, peripheral blood was drawn to determine MMP genotypes.
The study was approved by the ethics committee of our institution and written informed consent was obtained from all patients.
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Genetic study
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We extracted genomic DNA from white blood cells and determined the 1306C
T MMP-2 and 1171 5A
6A MMP-3 polymorphisms as previously described.12 A PCR-RFLP assay was used to determine the 1562C
T MMP-9 polymorphism using previously described PCR primers8 to generate a 435-bp amplicon that was further cleaved with the restriction endonuclease PaeI.
Follow-up
Clinical follow-up was carried out at outpatient visits or by contact with the general practitioner or the cardiologist. The primary endpoint of the study was cardiac mortality defined as cardiac death or urgent cardiac transplantation (United Network for Organ Sharing status 1 [UNOS1]).
Statistical analysis
We calculated mean values±SD for quantitative data. Differences in baseline characteristics in relation to genotypes were tested with the general linear model procedure for continuous variables and the
-test for categorical variables. LV function and exercise parameters were first compared between the three genotypes of each polymorphism. Then analyses were carried out after pooling heterozygotes with homozygotes for the rare allele; analyses were adjusted for age, gender, and cause of heart failure. We used KaplanMeier survival curves to assess late outcome. Hazard ratios and 95% CI were calculated using the Cox proportional hazard model. In these analyses, homozygotes for the rare allele (22) were pooled with heterozygotes (12). A unique model was used, which included age, gender, NYHA class, LVEF, and peak VO2 as adjustment variables. An interaction between aetiology (ischaemic vs. non-ischaemic) and genotype (11 vs. 12+22) was systematically included in the model. Whenever the interaction was statistically significant, further analyses were carried out separately in subjects with ischaemic or non-ischaemic aetiology. Two-sided test and
-values <0.05 were used to test for statistical significance. Statistical analyses were made using the SAS statistical package (release 6.12; SAS Institute, Cary, USA).
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Results
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Baseline characteristics
The baseline characteristics of the study population are listed in Table 1. Most patients were men with a mean age of 57±12 years. The cause of heart failure was ischaemic in 190 (43%) patients, non-ischaemic in 245 (55%) patients, and undetermined in 9 patients (2%). At entry for prognostic evaluation, 93% of the patients were on angiotensin-converting enzyme (ACE) inhibitor medication and 44% on beta-blocker treatment. Most of the patients not receiving beta-blocker at entry received a first dose of carvedilol or bisoprolol immediately after prognostic evaluation; thus 91% of the 444 patients were on beta-blocker therapy at discharge.
The distributions of the MMP-2, MMP-3, and MMP-9 genotypes are shown in Table 2; all allele distributions were in Hardy Weinberg equilibrium. Baseline clinical characteristics did not differ in relation to the MMP-2, MMP-3, and MMP-9 genotypes (Table 2).
LV function and exercise parameters
LV function parameters and exercise test variables were similar among MMP-2 genotypes. There was no evidence for any statistically significant difference in LV parameters between MMP-3 genotypes. There was, in contrast, a trend toward a higher peak VO2 in 5A/6A and 6A/6A patients compared to 5A/5A patients; when 6A allele carriers were compared to 5A/5A patients, these differences reached statistical significance. Finally, there was a trend toward lower LVEF and higher LVEDD in bearers of the T allele of MMP-9 polymorphism compared to CC patients; by contrast, exercise test variables did not differ between MMP-9 genotypes.
Survival analyses
Clinical follow-up was achieved for 443 patients; 1 patient was lost to follow-up. During a median follow-up of 717 days, there were 76 cardiac-related deaths, 12 heart transplantations, 4 of them urgent, and 9 deaths from non-cardiac causes. Since recent studies had reported different patterns of induction and possibly different roles of MMPs in human myocardium in ischaemic and non ischaemic HF, the survival analyses were carried out postulating a possible interaction between HF aetiology and MMP polymorphisms.
MMP-2 There was no evidence for any significant interaction between HF aetiology and MMP-2 polymorphism or any main effect of MMP-2 polymorphism on survival. Survival rates decreased similarly in CC homozygotes and in T-allele carriers (Fig. 1).

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Fig. 1 KaplanMeier curves of HF patients according to MMP-2 (1306C>T) genotypes (main effect; genotype: ns).
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MMP-3 There was a significant interaction between HF aetiology and MMP-3 polymorphism
, suggesting that MMP-3 polymorphism had a different effect on survival rate in patients with ischaemic and non-ischaemic HF. Survival curves (Fig. 2) indicated a better outcome in patients with non-ischaemic HF carrying the 5A/6A or 6A/6A genotypes
than in non-ischaemic patients with the 5A/5A genotype
or in ischaemic patients with any genotype
. Further multivariate analysis was carried out in non-ischaemic and ischaemic patients separately (Table 3). In patients with non-ischaemic cardiomyopathy, the MMP-3 5A/5A genotype was an independent predictor of cardiac mortality (HR 2.92;
); other variables retained in the model were LVEF
and peak VO2
. In patients with ischaemic cardiomyopathy, there was no evidence for any significant effect of MMP-3 genotype on survival; the two independent predictors of cardiac mortality were LVEF
and peak VO2
.
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Table 3 Impact of MMP-3 genotype on cardiac mortality according to heart failure aetiology: multivariate analysis
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MMP-9 There was no evidence for any significant interaction between HF aetiology and MMP-9 polymorphism in relation to survival. There was, in contrast, a statistically significant main effect of MMP-9 polymorphism on survival
. Patients carrying at least one T allele had lower survival rates than CC patients (Fig. 3). By multivariate analysis (Table 4), the presence of at least one T allele was an independent predictor of cardiac mortality (HR 1.81;
); other variables retained in the model were age
, NYHA class III or IV
, LVEF
, and peak VO2
.
Finally, to determine whether the two polymorphisms contribute independently to survival, we used a model in which both the MMP-3 and MMP-9 polymorphisms and an interaction term between aetiology and MMP-3 polymorphism were included. The results remain essentially the same. After adjustment for confounding variables, the HR of cardiac mortality was 1.8 (1.083.00) for MMP-9 (CT+TT vs. CC). Further analyses in non-ischaemic and ischaemic patients separately showed: (1) in non-ischaemic patients, a HR of 2.91 (1.276.55) for MMP-3 (5A5A vs. 5A6A+6A6A) and a HR of 1.8 (0.774.33) for MMP-9; (2) in ischaemic patients, a HR of 0.79 (0.371.66) for MMP-3 and a HR of 1.79 (0.943.42) for MMP-9.
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Discussion
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A large number of MMPS have been identified in human myocardium (for a review see1). The function of these enzymes is to catabolise proteins of the extracellular matrix. The activation of MMPs is implicated in LV remodelling and is potentially important in HF progression. increased MMP-2, MMP-3, and MMP-9 activities have been documented in myocardial samples from HF patients.5 In the present study, we analysed the possible influence of polymorphisms in the MMP-2 (1306C
T), MMP-3 (1171 5A
6A), and MMP-9 (1562C
T) gene promoters on clinical outcome of HF patients. Our findings indicate that MMP-3 polymorphism is specifically associated with cardiac mortality in patients with non-ischaemic HF, whereas MMP-9 polymorphism is associated with cardiac mortality independently of HF aetiology. In contrast, there was no evidence of any association between MMP-2 polymorphism and cardiac survival. These data suggest that variability at the genetic locus of the MMP-3 and MMP-9 genes may contribute, at least in part, to HF outcome.
The present study demonstrates an association between MMP genotypes and clinical outcome. The mechanism of this association has yet to be determined. However, it is tempting to speculate that this effect may be related to increased myocardial production of MMPs. Indeed, in vitro studies have demonstrated that the 1306C MMP-2, 1171 5A MMP-3, and 1562T MMP-9 alleles are associated with increased activity of their respective promoters.68 Cultured fibroblasts and vascular smooth muscle cells transfected with the 5A MMP-3 constructs expressed a 2-fold higher amount of reporter gene product as compared with the 6A MMP-3 allele.7 Similarly, transient transfection experiments showed that the 1562T MMP-9 allele had a 2-fold higher promoter activity than the 1562C allele.8 Recent studies have demonstrated that MMP-3 and MMP-9 polymorphisms are also functional in vivo. Blankenberg et al.9 have reported plasma levels of MMP-9 according to the 1562 C
T MMP-9 polymorphism in patients with coronary artery disease: the T allele was associated with increased MMP-9 levels. Medley et al.10 have reported in vivo gene and protein expression of MMP-3 in relation to the 1171 5A
6A polymorphism: in 5A homozygotes, gene expression was 4-fold higher
and in 6A homozygotes 2-fold lower
compared with heterozygotes. Differences in gene expression were associated with corresponding significant changes in MMP-3 protein levels. Thus, MMP polymorphisms might have a physiological impact on myocardium matrix proteolysis and cardiac remodelling. These hypotheses might explain the lower survival of HF patients bearing MMP-3 and MMP-9 mutated alleles.
The effect of the 5A/5A MMP-3 genotype was limited to non-ischaemic patients, while the MMP-9 T allele was associated with cardiac mortality irrespective of HF aetiology. These findings are in agreement with previous studies that have demonstrated differential induction of MMPs in the myocardium of ischaemic and non-ischaemic HF patients. For example, Spinale et al.5 demonstrated that MMP-3 levels are increased in cardiomyopathy of non-ischaemic origin but not in ischaemic cardiomyopathy. According to these findings, the possible regulatory role of MMP-3 polymorphism may be especially marked in non-ischaemic cardiomyopathy and its impact on the survival of HF patients may be more pronounced in patients with non-ischaemic HF. By contrast, MMP-9 levels are increased in both ischaemic and non-ischaemic cardiomyopathy, supporting our finding of an association independent of HF aetiology.
The strengths of the present study include a prospective design and complete characterisation of HF aetiology since 98% of the patients had coronary angiography. In addition, we performed a systematic prognostic evaluation at baseline which made it possible to adjust for possible confounders of cardiac events in multivariate analyses. However, since inflation of type 1 errors due to multiple testing is always possible, our results should be taken as hypothesis-generating rather than conclusive and further studies in different populations should be made. Our negative results with MMP-2 polymorphism should be interpreted in view of the statistical power of the study: with a power of 80% and a significance value of 0.05, the sample size of this study allowed us to detect a HR of 2 between T carriers and CC patients. Moreover, studies using intermediate phenotypes, such as MMP myocardial levels or sequential changes in LV volume, could help to clarify the relationship between MMP polymorphisms and HF progression.
In conclusion, our data suggest that MMP-3 and MMP-9 polymorphisms contribute, at least in part, to the variability in the occurrence of cardiac events in HF patients. These results may have important clinical implications. In the future, MMP genotyping may be used as a possible test for risk stratification of HF patients and eventually might help to identify patients who could benefit most from therapeutic strategies designed to inhibit MMP activity in the failing heart.
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Footnotes
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1 doi:10.1016/j.ehj.2004.02.025. 
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- Matrix metalloproteinase gene polymorphisms in heart failure: new pieces to the myocardial matrix puzzle
- Francis G Spinale
EHJ 2004 25: 631-633.
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