Improvement in collagen metabolism after 12 weeks cardiac resynchronization therapy in patients with ischaemic cardiomyopathy

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1 Research Report Improvement in collagen metabolism after 12 weeks cardiac resynchronization therapy in patients with ischaemic cardiomyopathy Journal of International Medical Research 41(1) ! The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalspermissions.nav DOI: / imr.sagepub.com Mingjiang Li, 1,2 Yanli Zhou, 1,2 Yongming Zhou, 1,2 Kamesh Babu 1,2 and Yueling Wang 1,2 Abstract Objectives: To investigate the effects of cardiac resynchronization therapy (CRT) on collagen metabolism biomarkers and their relationship to cardiac function, in patients with ischaemic cardiomyopathy (ICM). Methods: Serum levels of matrix metalloproteinase (MMP)-9, tissue inhibitor of MMP-9 (TIMP-1), carboxyterminal propeptide of type I procollagen (PICP) and carboxyterminal telopeptide of type I collagen (ICTP) were quantified before and after 12 weeks treatment, in patients with ICM receiving CRTand standard medical therapy (CRT group) or standard medical therapy alone (non- CRT group), and in controls. Cardiac function was measured echocardiographically. Results: MMP-9, TIMP-1, ICTP and the MMP-9/TIMP-1 ratio were significantly higher, and the PICP/ICTP ratio significantly lower, in patients with ICM (n ¼ 27) compared with controls (n ¼ 20). After 12 weeks treatment, MMP-9, TIMP-1, ICTP and the MMP-9/TIMP-1 ratio were significantly higher, and the PICP/ICTP ratio significantly lower, in the non-crt group (n ¼ 15) compared with the CRT group (n ¼ 12). The PICP/ICTP ratio correlated positively with TIMP-1 and negatively with MMP-9. The early/atrial ratio and left ventricular ejection fraction correlated positively and negatively, respectively, with the MMP-9/TIMP-1 ratio. Echocardiographic measurements of cardiac function were significantly worse in patients with ICM compared with controls and improved significantly after treatment in the CRT group. Conclusions: In ICM, collagen degradation biomarkers were elevated and correlated positively with cardiac function. CRT partially reversed the deterioration in collagen metabolism and enhanced cardiac function. 1 Cardiovascular Research Institute, Wuhan University, Wuhan, Hubei, China 2 Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei, China Corresponding author: Professor Mingjiang Li, Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei , China. mingjiangli@yahoo.com.cn

2 201 Li et al. Keywords Cardiac resynchronization therapy, collagen metabolism, ischaemic cardiomyopathy, matrix metalloproteinase Date received: 2 September 2012; accepted: 11 September 2012 Introduction Cardiac resynchronization therapy (CRT) is a valuable treatment for myocardial dysfunction occurring secondary to coronary artery occlusion, also known as ischaemic cardiomyopathy (ICM). 1 3 One of the major contributors to the myocardial fibrosis that develops in ICM is changes in the extracellular matrix (ECM). 4 6 Endogenous enzymes involved in ECM remodelling include the matrix metalloproteinases (MMPs), whose substrates are different types of collagen, and tissue inhibitors of MMPs (TIMPs). 7,8 Serum levels of MMP-9 and its specific inhibitor TIMP-1 are markers of extracellular collagen degradation and inhibition of collagen degradation, respectively. During collagen synthesis, carboxyterminal propeptide of type I procollagen (PICP) is cleaved from procollagen I and released into the blood, while during collagen degradation by MMP, carboxyterminal telopeptide of type I collagen (ICTP) is cleaved and released into the blood. Thus, PICP 9 and ICTP 10 can also be used as biomarkers of collagen synthesis and degradation, respectively. Research has shown an imbalance of MMP proteases and inhibitors in ICM, with activation of intracellular MMPs inducing increased levels of MMPs in the blood and tissues. 11 However, there have been few published reports on the effect of CRT on biomarkers of collagen metabolism in ICM. The present study investigated levels of MMP-9, TIMP-1, PICP and ICTP, together with the effect of CRT on collagen turnover, in patients with ICM. This study also explored the relationship between collagen metabolism and cardiac function, measured using echocardiography. Patients and methods Patients Patients with ICM, admitted to the Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei, China, were recruited sequentially to the study between June 2009 and December The diagnosis of ICM was based on the presence of all of the following criteria: (i) a positive stress test; (ii) a selective coronary angiogram (SCA) showing at least one vessel with >75% stenosis; (iii) a history of myocardial infarction; (iv) previous percutaneous transluminal coronary angioplasty; (v) cardiac failure classified as New York Heart Association stage 2, pulmonary congestion on chest X-ray, an early/atrial (E/A) ratio for diastolic ventricular filling velocities <1 and a mean left ventricular ejection fraction (LVEF) 40%; (vi) exclusion of other causes of heart failure. After initial evaluation, patients were randomly assigned (using a computer-generated randomization schedule) to either the CRT group or the non-crt group in a 4:5 ratio. The CRT group received CRT in addition to optimal standard medical therapy; the non-crt group received only optimal standard medical therapy. Normal age-matched healthy volunteers with no apparent myocardial infarction, valvular heart disease or hypertension, who had undergone various tests including SCA to exclude heart failure and coronary heart disease, were also recruited from the Department of Cardiology, Renmin Hospital of Wuhan University, as controls.

3 Journal of International Medical Research 41(1) 202 Exclusion criteria included all other conditions that could affect collagen metabolism (including pregnancy, hyperthyroidism, cancer and inflammation, other types of cardiomyopathy including dilated cardiomyopathy, rheumatic diseases, chronic hepatic disease, kidney failure, pulmonary fibrosis and treatment with corticosteroids). Patients in the CRT and non-crt groups were given optimal standard treatment for chronic heart failure throughout the study period, including angiotensinconverting enzyme inhibitors (ACEis), angiotensin-receptor blockers (ARBs), spironolactone, b-receptor blockers, positive inotropics, statins and aspirin. All study participants provided written informed consent. The study protocol was approved by the Medical Ethics Committee of Renmin Hospital of Wuhan University, Wuhan, Hubei, China. CRT Group Patients in the CRT group were implanted with CRT pacemakers (Frontier TM II model 5596, St Jude Medical, Sylmar, CA, USA) according to standard procedures. Biochemical Analyses Eearly in the morning at the beginning of the study and after 12 weeks treatment, fasting 4-ml peripheral venous blood samples were taken from patients in the CRT and non- CRT groups. A single fasting peripheral venous sample was also taken from each subject in the control group. All samples were centrifuged at g and 4 C for 10 min. The resulting serum was then divided into four equal parts, and stored at 80 C until analysis. Serum MMP-9 and TIMP-1 concentrations were measured using enzyme-linked immunosorbent assay (ELISA) kits (Amersham Pharmacia Biotech, GE Healthcare Life Sciences, Shanghai, China) in accordance with the manufacturer s instructions. Serum PICP and ICTP were measured using radioimmunoassay kits (Haiyan Medical Biotechnology Centre, Shanghai, China) in accordance with the manufacturer s instructions. All assays were performed in duplicate. Echocardiography Two-dimensional and Doppler echocardiography (HP Õ Sonos 5500, Hewlett- Packard/Philips Healthcare, Amsterdam, The Netherlands) was performed in patients with ICM before and after the 12-week treatment period, and in controls by echocardiographers blinded to each participant s information. Measurements taken included the left ventricular end-diastolic diameter corrected for body surface area (LVEDD BSA), E/A ratio and LVEF; results recorded were an average of three readings taken over five continuous cardiac cycles. Statistical Analyses Data were presented as mean SD. Intergroup comparisons of continuous variables were made using a two-group t-test; intragroup comparisons were made using a paired t-test. Correlation analyses were performed using Spearman s rank correlation. A P-value of <0.05 was considered to be statistically significant. All statistical analyses were performed using SPSS Õ software, version 13.0 (SPSS Inc., Chicago, IL, USA). Results A total of 27 patients with ICM (21 males and 6 females; mean age years, range years) were recruited to the study and divided as outlined above, into a CRT group (n ¼ 12) and a non-crt group (n ¼ 15). There were no statistically significant differences between the groups in terms of age, sex, low-density lipoprotein level,

4 203 Li et al. presence of hypertension or history of diabetes mellitus (data not shown). A further 20 healthy subjects (13 males and 7 females; mean age years, range years) were recruited as controls. Measurements of MMP-9 and TIMP-1 made using ELISAs had intragroup coefficient variations of 4.1% and 4.5%, respectively. Serum PICP and ICTP levels (measured using radioimmunoassays) had intragroup coefficient variations of 4.2% and 4.8%, respectively. Collagen Metabolism Levels of serum collagen metabolism markers in ICM patients and control subjects are shown in Table 1. MMP-9 (P < 0.01), TIMP-1 (P < 0.01), the MMP-9/TIMP-1 ratio (P < 0.01) and ICTP (P < 0.05) were significantly higher in ICM patients than in controls. The PICP/ICTP ratio was significantly lower in ICM patients than in controls (P < 0.01). There were no significant differences in PICP levels between the groups. Levels of collagen metabolism markers in the two groups of ICM patients before and after 12 weeks treatment are given in Table 2. There were no significant differences in the levels of biomarkers between the two groups before treatment. After 12 weeks treatment, the CRT group showed significant reductions in MMP-9, TIMP-1 and ICTP compared with pretreatment levels (all P < 0.01), while the non- CRT group showed significant reductions in only MMP-9 and ICTP levels (P < 0.05). In addition, there was a significant reduction in the MMP-9/TIMP-1 ratio and a significant increase in the PICP/ICTP ratio (both P < 0.01) after 12 weeks treatment in the CRT group, but not in the non-crt group. After 12 weeks treatment, MMP-9, TIMP-1, ICTP and the MMP-9/TIMP-1 ratio were significantly higher, and the PICP/ICTP ratio was significantly lower, in the non-crt group compared with the CRT group (P < 0.05 for all) (Table 2). Analysis of correlations within the posttreatment levels of various collagen metabolism markers in ICM patients showed positive correlations between MMP-9 and ICTP (r ¼ 0.301, P ¼ 0.012), TIMP-1 and PICP (r ¼ 0.786, P ¼ 0.041), and the PICP/ ICTP ratio and TIMP-1 (r ¼ 0.673, P ¼ ), and negative correlations between the PICP/ICTP ratio and MMP-9 (r ¼ 0.458, P ¼ ) and the MMP-9/ TIMP-1 and PICP/ICTP ratios (r ¼ 0.639, P ¼ 0.043). Table 1. Serum collagen metabolism markers in patients with ischaemic cardiomyopathy (ICM) and controls. Marker ICM patients n ¼ 27 Controls n ¼ 20 MMP-9, ng/l b TIMP-1, ng/l b MMP-9/TIMP-1 ratio b PICP, mg/l ICTP, mg/l a PICP/ICTP ratio b Data presented as mean SD. MMP-9, matrix metalloproteinase-9; TIMP-1, tissue inhibitor of MMP-9; PICP, carboxyterminal propeptide of type I procollagen; ICTP, carboxyterminal telopeptide of type I collagen. a P < 0.05 and b P < 0.01 compared with controls using two-group t-test.

5 Journal of International Medical Research 41(1) 204 Cardiac Function The E/A ratio ( versus , P < 0.01) and LVEF ( % versus %, P < 0.01) were significantly lower, and the LVEDD BSA ( mm versus mm, P < 0.01) significantly higher, in ICM patients compared with controls. After 12 weeks treatment, LVEDD BSA ( mm versus mm, P < 0.05), LVEF (42 9.3% versus %, P < 0.01) and E/A ratio values ( versus , P < 0.01) were significantly improved in the CRT group compared with pretreatment, but no significant differences were seen after treatment in the non-crt group ( mm versus mm, % versus % and versus , respectively). Before CRT implantation, parameters representing left ventricular diastolic function in patients with ICM were significantly correlated with collagen metabolism biomarkers: there was a significant correlation between the E/A ratio and the MMP-9/ TIMP-1 ratio (r ¼ 0.712, P ¼ 0.018) and between the LVEDD BSA and PICP and ICTP (r ¼ 0.578, P ¼ and r ¼ 0.350, P ¼ 0.020, respectively). However, the E/A ratio and LVEDD BSA did not show any correlation with the PICP/ICTP ratio. In contrast, there was a negative correlation between LVEF and the MMP-9/TIMP-1 ratio (r ¼ 0.67, P ¼ 0.036). After 12 weeks treatment, there was a negative correlation between the LVEDD BSA and the PICP/ICTP ratio (r ¼ 0.823, P ¼ 0.032) in the CRT group. Discussion Ischaemic cardiomyopathy is a concept popularized by Burch et al. in that refers to myocardial dysfunction secondary to coronary artery occlusion. Microscopically, there is increased Table 2. Serum collagen metabolism markers in patients with ischaemic cardiomyopathy, receiving cardiac resynchronization therapy and standard medical therapy (CRT group) or standard medical therapy alone (non- CRT group). CRT group Non-CRT group Marker Pretreatment After 12 weeks treatment Pretreatment After 12 weeks treatment MMP-9, ng/l b a,c TIMP-1, ng/l b c MMP-9/TIMP-1 ratio b c PICP, mg/l ICTP, mg/l b a,c PICP/ICTP ratio b c Data presented as mean SD. MMP-9, matrix metalloproteinase-9; TIMP-1, tissue inhibitor of MMP-9; PICP, carboxyterminal propeptide of type I procollagen; ICTP, carboxyterminal telopeptide of type I collagen. a P < 0.05 and b P < 0.01 compared with pretreatment values using paired t-test. c P < 0.05 compared with CRT group using two-group t-test.

6 205 Li et al. interstitial fibrosis replacing the normal intercellular structure. 4 Collagen is the main component of ECM and maintains cellular shape and arrangement, as well as transmitting signals. 13 When the myocardium is in an ischaemic state, the structure of the collagen network and the ratios of the different types of collagen appear to change. 4,6 Human and animal studies have suggested that regulating the composition of ECM can provide new insights into the prevention of cardiac remodelling in ischaemia Invasive myocardial biopsy has been the gold standard for evaluating myocardial fibrosis and tissue repair. However, noninvasive ways of assessing collagen metabolism (by measuring the biomarkers MMP and TIMP) have been developed and may replace myocardial biopsy. 6,10,11,17 19 In the present study, MMP-9 and its inhibitor TIMP-1 were chosen as markers of collagen haemostasis in ischaemic myocardium. 20 Due to the antagonistic relationship between MMP-9 and TIMP-1, the ratio of MMP-9 to TIMP-1 can reflect MMP hydrolyzing ability and activity, and can also indirectly estimate the balance between collagen synthesis and degradation. Similarly, the ratio of PICP to ICTP can be used to reflect the balance between type I collagen synthesis and degradation. It is known that any imbalance between a collagenase and its endogenous inhibitor can lead to changes in the ECM, causing tissue remodelling. 5,6,10,14,18,21 In the present study, MMP-9 and TIMP-1 levels and the MMP- 9/TIMP-1 ratio were increased in ICM patients compared with normal control subjects, suggesting that type I collagen degradation is higher than synthesis in ischaemic myocardium. In addition, ICTP, which is the metabolite of type I collagen degradation, was also significantly increased in patients compared with normal control subjects. These results are similar to those reported from a small study by Wilson et al. 21 The positive correlation between MMP-9 and ICTP demonstrated in the present study suggests that MMP-9 plays an important role in ECM degradation in ICM patients. Interestingly, TIMP-1 and MMP-9 levels were both increased, with the latter increasing more than the former; the rise in MMP-9 activity may cause an increase in TIMP-1 as a result of negative feedback, to compensate for increased type I collagen degradation. 10 In the present study, ICTP levels were positively correlated with MMP-9 levels, which were higher than TIMP-1 levels, giving rise to high collagen degradation. The echocardiographic measurements from the present study indicated that diastolic function (the E/A ratio) was decreased in ICM patients compared with controls. ACEi/ARB and spironolactone therapy have been shown to partly reverse the abnormal metabolism of cardiac collagen tissue and enhance cardiac function in ICM patients. 16,19,22 24 Improvements in haemodynamics and electromechanical synchronization after CRT appear to be related to corrected collagen metabolism. 25,26 However, no clinical observations of the effect of CRT on collagen metabolism in ICM have previously been reported. The results of the present study confirm that there is reduced cardiac compliance in ICM, causing diastolic dysfunction; however, it is not clear why systolic function is also affected. Degradation of type I collagen, which is harder and stiffer than type III collagen, appears to increase. 27 In conclusion, the present study investigated collagen metabolism in patients with ICM and demonstrated that serum collagen metabolism biomarkers may be useful in assessing the extent of fibrosis and cardiac dysfunction. Disordered collagen turnover and cardiac dysfunction were improved by CRT, given in addition to optimal standard treatment for chronic heart failure.

7 Journal of International Medical Research 41(1) 206 Acknowledgements The authors are grateful to the other staff members at their institutions for their valuable comments. Declaration of Conflicting Interest The authors declare that there are no conflicts of interest. Funding This study was financially supported by the National Natural Science Grant of China (No ). References 1. Bank AJ, Burns KV, Gage RM, et al. Cardiac resynchronization therapy in the real world: comparison with the COMPANION study. J Card Fail 2012; 18: Stevenson WG, Hernandez AF, Carson PE, et al. Indications for cardiac resynchronization therapy: 2011 update from the Heart Failure Society of America Guideline Committee. J Card Fail 2012; 18: Niederer SA, Lamata P, Plank G, et al. Analyses of the redistribution of work following cardiac resynchronisation therapy in a patient specific model. PLoS One 2012; 7: e Mukherjee D and Sen S. Alteration of collagen phenotypes in ischemic cardiomyopathy. J Clin Invest 1991; 88: Tyagi SC, Kumar SG, Hass SJ, et al. Posttranscriptional regulation of extracellular matrix metalloproteinase in human heart endstage failure secondary to ischemic cardiomyopathy. J Mol Cell Cardiol 1996; 28: Schwartzkopff B, Fassbach M, Pelzer B, et al. Elevated serum markers of collagen degradation in patients with mild to moderate dilated cardiomyopathy. Eur J Heart Fail 2002; 4: Fan D, Takawale A, Lee J, et al. Cardiac fibroblasts, fibrosis and extracellular matrix remodeling in heart disease. Fibrogenesis Tissue Repair 2012; 5: Krenning G, Zeisberg EM and Kalluri R. The origin of fibroblasts and mechanism of cardiac fibrosis. J Cell Physiol 2010; 235: Kosmala W, Przewlocka-Kosmala M, Wojnalowicz A, et al. Integrated backscatter as a fibrosis marker in the metabolic syndrome: association with biochemical evidence of fibrosis and left ventricular dysfunction. Eur Heart J Cardiovasc Imaging 2012; 13: Lombardi R, Betocchi S, Losi MA, et al. Myocardial collagen turnover in hypertrophic cardiomyopathy. Circulation 2003; 108: Blankenberg S, Rupprecht HJ, Poirier O, et al. Plasma concentrations and genetic variation of matrix metalloproteinase 9 and prognosis of patients with cardiovascular disease. Circulation 2003; 107: Burch GE, Giles TD and Colcolough HL. Ischemic cardiomyopathy. Am Heart J 1970; 79: Hein S and Schaper J. The extracellular matrix in normal and diseased myocardium. J Nucl Cardiol 2001; 8: Dı ez J, Querejeta R, Lo pez B, et al. Losartan-dependent regression of myocardial fibrosis is associated with reduction of left ventricular chamber stiffness in hypertensive patients. Circulation 2002; 105: Li YY, Feng Y, McTiernan CF, et al. Downregulation of matrix metalloproteinases and reduction in collagen damage in the failing human heart after support with left ventricular assist devices. Circulation 2001; 104: Wang JM, Wang Y, Zhu ZS, et al. Diverse effects of long-term treatment with imidapril and irbesartan on cell growth signal, apoptosis and collagen type I expression in the left ventricle of spontaneously hypertensive rats. Life Sci 2004; 75: Spinale FG, Coker MJ, Heung LJ, et al. A matrix metalloproteinase induction/activation system exists in the human left

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