Changes in matrix metalloproteinase-9 levels during progression of atrial fibrillation

Abstract

Objectives:

To observe levels of matrix metalloproteinase (MMP)-9 and evaluate their significance in various stages of idiopathic atrial fibrillation (AF).

Methods:

Patients with idiopathic AF were recruited into this prospective study and classified into one of three groups according to stage of disease progression: paroxysmal AF; persistent AF; permanent AF. Healthy individuals were enrolled as control subjects. Serum levels of MMP-9 in all four groups were determined using a double-antibody sandwich enzyme-linked immunosorbent assay.

Results:

Each AF group included 25 patients; 40 healthy individuals were included as controls. MMP-9 levels in the three AF groups were significantly higher than in the control group: 168.72 ± 25.970, 201.36 ± 31.26 and 253.20 ± 22.99 ng/ml for the paroxysmal, persistent and permanent AF groups respectively, versus 76.80 ± 14.90 ng/ml for the control group. MMP-9 levels increased with idiopathic AF disease progression (P < 0.05).

Conclusions:

An elevated MMP-9 level appears to be associated with a diagnosis of AF. MMP-9 levels appear to increase in relation to the stage of idiopathic AF progression.

Keywords

Atrial fibrillation concentration disease progression extracellular matrix matrix metalloproteinase-9 pathological mechanism

Introduction

Atrial fibrillation (AF) is a frequently occurring disease that is encountered in emergency medicine settings. Experimental and clinical research have shown that, whatever the initial cause or trigger, there is a relationship between AF and alterations in atrial electrical properties.¹ However, the pathogenesis of AF is not yet fully understood. Atrial interstitial tissue fibrosis is an important mechanism of AF^2,3 that could lead to atrium diameter expansion, atrial wall thinning and, thus, atrium structure reconstruction.⁴ Endogenous enzymes involved in extracellular matrix remodelling include the matrix metalloproteinases (MMPs), whose substrates are different types of collagen; consequently, the serum level of MMP-9 is considered to be a marker of extracellular collagen degradation.⁵ Studies have found that MMPs may be implicated in the development of myocardial fibrosis. For example, the MMP-9 level is an important index for myocardial fibrosis,^6,7 and also has a close relationship with other cardiovascular diseases.^8,9 The present study observed MMP-9 levels in patients with idiopathic AF at different stages of disease progression, compared these levels with those found in healthy controls, and assessed the relationship between MMP-9 levels and idiopathic AF progression.

Patients and methods

Participants

This study recruited people with idiopathic AF who were outpatients or inpatients receiving care at Renmin Hospital of Wuhan University, Wuhan, Hubei, China between July 2010 and July 2012. Patients were recruited sequentially and were allocated to one of three study groups according to stage of idiopathic AF disease progression (paroxysmal AF, persistent AF and permanent AF), based on the American College of Cardiology/American Heart Association/European Society of Cardiology 2006 guidelines for AF treatment,¹⁰ until there were 25 patients in each group. Patients included in the study were free from organic cardiovascular disease, hyperthyroidism, pregnancy and infectious diseases, and had not undergone surgery within the previous 2 months. Echocardiography was performed to confirm that patients did not have atrial thrombus. None of the patients had undergone electrical or pharmacological cardioversion therapy in the previous month or had taken angiotensin-converting enzyme inhibitors, corticosteroids or statins in the previous week. Patients with AF who also had structural heart disease were excluded from the study.

The control group comprised people attending the hospital for routine physical examination who exhibited no special discomfort on examination or any abnormality on medical, blood biochemistry, electrocardiogram, echocardiographic (including liver and gallbladder echocardiographic), spleen or kidney ultrasonographic, or chest X-radiographic examinations.

The study protocol was approved by the Medical Ethics Committee of Renmin Hospital of Wuhan University, Wuhan, Hubei, China. All study participants provided written informed consent.

Blood sampling and analysis

Early in the morning after enrolment, 4-ml venous blood samples were taken from each patient in the fasting state. After treatment with 100 g/l sodium citrate as anticoagulant and centrifugation (4-16K, Sigma-Aldrich, Germany) at 1360 g for 10 min at 4℃, serum was dispensed into EP tubes (MCT-150-C, Axygen, America) and stored at −80℃ until analysis. Blood samples were analysed in batches every 3 months. The serum concentration of MMP-9 was determined using a double-antibody sandwich enzyme-linked immunosorbent assay kit (Shanghai Senxiong Biotech, Shanghai, China), according to the manufacturer’s instructions.

Additional assessments

Body mass index, left atrial diameter, left ventricular ejection fraction, systolic and diastolic blood pressure, and concentrations of serum Na+ and K+ were determined by standard methods after enrolment.

Statistical analyses

This study recruited the same number of patients into each group to help with statistical comparisons. Continuous data were presented as mean ± SD; categorical data were presented as n. Statistical comparisons were undertaken using SPSS® software, version 11.0 (SPSS Inc., Chicago, IL, USA). A P-value < 0.05 indicated statistical significance. Between-group comparisons of continuous variables were undertaken using independent-samples t-test.

Results

This study enrolled 75 patients with AF: 25 cases for each stage of idiopathic AF disease progression (paroxysmal AF, persistent AF, permanent AF) and 40 healthy control subjects. There were no significant between-group differences in age, sex, body mass index, left atrial diameter, left ventricular ejection fraction, blood pressure, or serum Na⁺ and K⁺ concentrations (Table 1).

Table 1.

Baseline characteristics of patients with different stages of idiopathic atrial fibrillation (AF) and healthy controls, enrolled in a study investigating matrix metalloproteinase-9 levels and their relationship with AF progression.

Characteristic	Paroxysmal AF, n = 25	Persistent AF, n = 25	Permanent AF, n = 25	Controls, n = 40
Age, years	56.1 ± 17.3	58.6 ± 15.1	59.1 ± 16.4	54.8 ± 14.9
Sex, male/female	19/6	20/5	19/6	31/9
BMI, kg/m²	27 ± 3.6	29 ± 4.6	28 ± 5.1	27 ± 5.7
Left atrial diameter, mm	28.3 ± 6.7	30.1 ± 5.8	26.4 ± 7.2	26.1 ± 8.7
LVEF, %	64 ± 11	66 ± 11	64 ± 13	69 ± 9
Systolic BP, mmHg	125.2 ± 12.1	126.3 ± 10.8	122.3 ± 12.4	117.6 ± 13.6
Diastolic BP, mmHg	78.0 ± 11.9	76.0 ± 8.1	78.0 ± 10.1	77.2 ± 11.8
Serum Na⁺, mmol/l	142.5 ± 7.4	140.5 ± 6.2	143.5 ± 8.9	143.1 ± 8.1
Serum K⁺, mmol/l	4.2 ± 0.7	4.3 ± 0.6	4.3 ± 0.7	4.4 ± 0.8

Data presented as mean ± SD or n.

BMI, body mass index; BP, blood pressure; LVEF, left ventricular ejection fraction.

No statistically significant differences observed (P ≥ 0.05; independent-samples t-test).

Levels of MMP-9 in the three AF-groups were significantly higher than in the control group (P < 0.01) and showed a significant gradual increase from paroxysmal AF through persistent AF to permanent AF (P < 0.05) (Table 2).

Table 2.

Comparison of matrix metalloproteinase (MMP)-9 levels in patients with different stages of idiopathic atrial fibrillation (AF) and healthy controls.

Characteristic	Paroxysmal AF, n = 25	Persistent AF, n = 25	Permanent AF, n = 25	Controls, n = 40
MMP-9 (ng/ml)	168.72 ± 25.97^a,b	201.36 ± 31.26^a,b	253.20 ± 22.99^a,b	76.80 ± 14.90

Data presented as mean ± SD.

P < 0.05 for between-group treatment comparisons (independent-samples t-test).

P < 0.01 compared with control group (independent-samples t-test).

Discussion

Under physiological conditions, the extracellular matrix is in homeostasis by being continuously produced and degraded, and an imbalance in its synthesis or degradation is related to the occurrence of cardiovascular disease.^11,12 MMPs can degrade many proteins of the extracellular matrix, such as collagen, laminin, fibronectin, proteoglycans and elastin.^13–15 Increases in expression and activity of MMPs are observed in the development of many cardiovascular diseases. Specifically, MMP-9 has been implicated in the process of myocardial fibrosis.^16,17 In addition, MMP-9 was shown to be induced – and generated collagen-matrix fragments, such as endostatin and angiostatin – in patients undergoing coronary artery bypass grafting.¹⁸ In murine studies, Ducharme and colleagues¹⁹ found that MMP-9 played a key role in extracellular remodelling after myocardial infarction: following infarction, the loss of MMP-9 activity in mice with targeted deletion of mmp9 led to a significant reduction in collagen deposition in the infracted region. In addition, a reduction in inflammatory cell infiltration was observed in the mmp9-knockout mice, and the authors concluded that left ventricular enlargement would ease after MI, thus providing evidence that MMP-9 is associated with cardiac remodelling.¹⁹ In the present study we observed the relationship between MMP-9 levels and the occurrence and progression of AF.

We selected patients with idiopathic AF as research participants to exclude the influence of organic diseases on MMP-9 levels. Close relationships between MMP levels and AF have been reported.^20–22 Patients with both chronic rheumatic heart disease and AF showed abnormal expression of MMP-1,²³ and MMP-9 has been associated with rapid atrial arrhythmia.^24,25 In an animal model of rapid atrial pacing-induced atrial failure, MMP-9 activity in the atrial intramuscular matrix increased by 50% compared with sinus rhythm, while levels of specific tissue inhibitors decreased by 50%.²⁶ Another study has shown that MMP inhibitors can decrease left ventricular dilatation and wall thinning of heart failure induced by overdrive pacing, to reduce atrial chamber wall stress.²⁷ However, there is no report on whether the MMP-9 level is related to AF development and progression in humans.

We found that MMP-9 levels in patients with idiopathic AF were significantly higher those in the healthy control population, indicating that MMP-9 may play a role in the occurrence and maintenance of AF. MMP-9 may be involved in degradation and reconstruction of the extracellular matrix under the influence of metallic zinc ions: it could, for example, cut off extracellular matrix components, regulate cell adhesion, act on extracellular components or other protein components and activate potential biological function, and participate directly or indirectly in tissue remodelling and wound repair.²⁸ When MMP-9 levels are elevated, the above-mentioned functions are enhanced, which would result in excessive extracellular matrix degradation, increased tissue remodelling and myocardial fibrosis, and could contribute to micro re-entry and eventually lead to AF.²⁹

Our study also found that MMP-9 levels gradually increased from paroxysmal AF through persistent AF to permanent AF. In addition, we identified significant differences in MMP-9 levels between the three study groups. Other research has demonstrated that AF is characterized by self-sustaining and gradual progression, and that the majority of cases of paroxysmal AF will eventually evolve into persistent or even permanent fibrillation.³⁰ The mechanism for this is not very clear. Other studies have mainly focused on the electrical remodelling of AF, believing that ion channel remodelling mainly causes AF progression.^31,32 The results of the present study suggest that a gradual elevation in the MMP-9 level may worsen myocardial interstitial fibrosis, and that structural remodelling may also play an important role in AF progression, at least in part due to the known involvement of MMP-9 in the structural remodeling process.^26,27

The limitation of the present study is its sample size; a larger sample is needed for further research. Given that AF is a multifactorial disease of unclear aetiology, further research should also investigate factors in addition to MMP-9 that may have a bearing on its development and course.

In conclusion, our study found that MMP-9 levels are higher in patients with AF than in healthy controls, and that MMP-9 may therefore be associated with the development of AF. In addition, our findings provide indications of a relationship between the increase in the MMP-9 level and the progression of AF. Further research is necessary.

Footnotes

Declaration of conflicting interest

The authors declare that there are no conflicts of interest.

Funding

This study was supported by the National Natural Science Foundation of China (no. 81170085).

References

Kabra

Heist

Barrett

. Incidence and electrophysiologic properties of dissociated pulmonary vein activity following pulmonary vein isolation during catheter ablation of atrial fibrillation. J Cardiovasc Electrophysiol 2010; 21: 1338–1343.

Takahashi

Kume

Wakisaka

. Novel strategy to prevent atrial fibrosis and fibrillation. Circ J 2012; 76: 2318–2326.

Liu

. Hyperglycemia aggravates atrial interstitial fibrosis, ionic remodeling and vulnerability to atrial fibrillation in diabetic rabbits. Anadolu Kardiyol Derg 2012; 12: 543–550.

Kostin

Klein

Szalay

. Structural correlate of atrial fibrillation in human patients. Cardiovasc Res 2002; 54: 361–379.

Veidal

Nielsen

Leeming

. Phosphodiesterase inhibition mediates matrix metalloproteinase activity and the level of collagen degradation fragments in a liver fibrosis ex vivo rat model. BMC Res Notes 2012; 5: 686–686.

Fan

Takawale

Lee

. Cardiac fibroblasts, fibrosis and extracellular matrix remodeling in heart disease. Fibrogenesis Tissue Repair 2012; 5: 15–15.

Georgescu

Aronovitz

Iovanna

. Decreased metalloprotease 9 induction, cardiac fibrosis, and higher autophagy after pressure overload in mice lacking the transcriptional regulator p8. Am J Physiol Cell Physiol 2011; 301: C1046–C1056.

Spinale

Coker

Heung

. A matrix metalloproteinase induction/activation system exists in the human left ventricular myocardium and is upregulated in heart failure. Circulation 2000; 102: 1944–1949.

Renko

Kalela

Jaakkola

. Serum matrix metalloproteinase-9 is elevated in men with a history of myocardial infarction. Scand J Clin Lab Invest 2004; 64: 255–261.

10.

Fuster

Rydén

Cannom

. ACC/AHA/ESC 2006 Guidelines for the management of patients with atrial fibrillation. Circulation 2006; 114: 700–752.

11.

Spinale

. Matrix metalloproteinases: regulation and dysregulation in the failing heart. Circ Res 2002; 90: 520–530.

12.

Scholze

Bladbjerg

Sidelmann

. Plasma concentrations of extracellular matrix protein fibulin-1 are related to cardiovascular risk markers in chronic kidney disease and diabetes. Cardiovasc Diabetol 2013; 12: 6–6.

13.

Vilen

Salo

Sorsa

. Fluctuating roles of matrix metalloproteinase-9 in oral squamous cell carcinoma. Sci World J 2013; 2013: 1–11.

14.

Kwak

Kim

Joshi

. Exercise training reduces fibrosis and matrix metalloproteinase dysregulation in the aging rat heart. FASEB J 2011; 25: 1106–1117.

15.

Zhang

. Exogenously administered secreted frizzled related protein 2 (Sfrp2) reduces fibrosis and improves cardiac function in a rat model of myocardial infarction. Proc Natl Acad Sci U S A 2010; 107: 21110–21115.

16.

Zervoudaki

Economou

Stefanadis

. Plasma levels of active extracellular matrix metalloproteinases 2 and 9 in patients with essential hypertension before and after antihypertensive treatment. J Hum Hypertens 2003; 17: 119–124.

17.

Iwanaga

Aoyama

Kihara

. Excessive activation of matrix metalloproteinases coincides with left ventrieular remodeling during transition from hypertrophy to heart failure in hypertensive rats. J Am Coll Cardiol 2002; 39: 1384–1391.

18.

Sodha

Clements

Boodhwani

. Endostatin and angiostatin are increased in diabetic patients with coronary artery disease and associated with impaired coronary collateral formation. Am J Physiol Heart Circ Physiol 2009; 296: H428–H434.

19.

Ducharme

Frantz

Aikawa

. Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J Clin Invest 2000; 106: 55–62.

20.

Wang

Yang

. [Role of matrix metalloproteinase and tissue inhibitor of metalloproteinase in atrial structural remodeling in patients with atrial fibrillation]. Nan Fang Yi Ke Da Xue Xue Bao 2010; 30: 1160–1162.

21.

Gan

Tang

. Differential expression of collagen and matrix metalloproteinases between left and right atria in patients with chronic atrial fibrillation. Sheng Li Xue Bao 2009; 61: 211–216. In Chinese, English abstract.

22.

Anné

Willems

Roskams

. Matrix metalloproteinases and atrial remodeling in patients with mitral valve disease and atrial fibrillation. Cardiovasc Res 2005; 67: 655–666.

23.

Marín

Roldán

Climent

. Is thrombogenesis in atrial fibrillation related to matrix metalloproteinase-1 and its inhibitor, TIMP-1? Stroke 2003; 34: 1181–1186.

24.

Kato

Fujimaki

Yoshida

. Impact of matrix metalloproteinase-2 levels on long-term outcome following pharmacological or electrical cardioversion in patients with atrial fibrillation. Europace 2009; 11: 332–337.

25.

Laurent

Moe

. Experimental studies of atrial fibrillation: a comparison of two pacing models. Am J Physiol Heart Circ Physiol 2008; 294: H1206–H1215.

26.

Hoit

Takeishi

Cox

. Remodeling of the left atrium in pacing-induced atrial cardiomyopathy. Mol Cell Biochem 2002; 238: 145–150.

27.

Dorian

Mangat

. Quality of life variables in the selection of rate versus rhythm control in patients with atrial fibrillation: observations from the Canadian Trial of Atrial Fibrillation. Card Electrophysiol Rev 2003; 7: 276–279.

28.

Santos

de Souza

Gerlach

. Inhibition of human pulpal gelatinases (MMP-2 and MMP-9) by zinc oxide cements. J Oral Rehabil 2004; 31: 660–664.

29.

Anné

Willems

Roskams

. Matrix metalloproteinases and atrial remodeling in patients with mitral valve disease and atrial fibrillation. Cardiovasc Res 2005; 67: 655–666.

30.

Chiang

Naditch-Brûlé

Murin

. Distribution and risk profile of paroxysmal, persistent, and permanent atrial fibrillation in routine clinical practice: insight from the real-life global survey evaluating patients with atrial fibrillation international registry. Circ Arrhythm Electrophysiol 2012; 5: 632–639.

31.

Huang

. Transient outward potassium current remodeling of human atrial myocytes during the development of atrial fibrillation. Chin J Cardiac Arrhyth 2005; 9: 188–190.

32.

Huang

Tang

. Changes of IK, ATP current density and allosteric modulation during chronic atrial fibrillation. Chin Med J 2005; 118: 1161–1166.