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Serum markers of deranged myocardial collagen turnover: Their relation to malignant ventricular arrhythmias in cardioverter-defibrillator recipients with heart failure
Congestive Heart Failure
Serum markers of deranged myocardial collagen turnover: Their relation to malignant ventricular arrhythmias in cardioverter-defibrillator recipients with heart failure Panayota Flevari, MD, a George Theodorakis, MD, b Dionyssios Leftheriotis, MD, a Christos Kroupis, MD, c Fotis Kolokathis, MD, a Kleanthi Dima, MD, c Maria Anastasiou-Nana, MD, a and Dimitrios Kremastinos, MD a Athens, Greece
Background Pathologic collagen remodeling has been involved in the occurrence of ventricular arrhythmias and sudden cardiac death in heart failure. The aim of the study was to investigate the relationship between malignant ventricular arrhythmias and cardiac collagen turnover indexes, expressing specific types of derangement in collagen physiology, in stable patients with an implantable cardioverter-defibrillator (ICD). Methods Seventy-four patients with an ICD and heart failure were studied. They had coronary artery disease (n = 42) or dilated cardiomyopathy, New York Heart Association classes I and II, and left ventricular ejection fraction 29% ± 1%. An ICD had been implanted for secondary (n = 36) or primary prevention of sudden cardiac death. We assessed (1) markers of collagen types I and III synthesis and their ratio: procollagen type I carboxyterminal peptide (PICP), procollagen type III aminoterminal peptide (PIIINP), and PICP/PIIINP; (2) markers of collagen degradation, degradation inhibition, and their ratio: matrix metalloproteinase 9 (MMP-9), tissue inhibitor of matrix metalloproteinase (TIMP) 1 (TIMP-1), and MMP-9/TIMP-1. Patients were prospectively followed up for 1 year. The number of episodes necessitating appropriate interventions for ventricular tachyarrhythmias (N170 beat/min) was related to the assessed parameters. Results
Multivariate analysis revealed a significant relation between the number of tachyarrhythmic episodes and MMP9/TIMP-1 (P = .007), PICP/PIIINP (P = .007), and ejection fraction (P = .04). No other significant relation was observed between arrhythmias and the remaining parameters.
Conclusion In heart failure, biochemical markers indicative of a deranged equilirium in myocardial collagen deposition/degradation and collagen I/III synthesis are related to ventricular arrhythmogenesis. Further studies are needed to investigate their predictive ability. (Am Heart J 2012;164:530-7.)
The prevention of sudden cardiac death (SCD) among patients with heart failure remains suboptimal. 1 The use of implantable cardioverter-defibrillators (ICDs) is practically the most efficacious therapy, although it is expensive and not without side effects. 2-4 Changes in cardiac extracellular matrix characterize the remodeling process in heart failure, leading to fibrosis formation, collagen alteration, and degradation. Collagen synthesis and degradation result from a From the a2nd Department of Cardiology, Attikon University Hospital, Athens, Greece, b Department of Cardiology, Henry Dunant Hospital, Athens, Greece, and cBiochemical Laboratory, Attikon University Hospital, Athens, Greece. Submitted March 2, 2012; accepted July 13, 2012. Reprint requests: Panayota Flevari, MD, 2nd Department of Cardiology, Attikon University Hospital, Rimini 1, 12402 Haidari, Athens, Greece. E-mail: [email protected] 0002-8703/$ - see front matter © 2012, Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/j.ahj.2012.07.006
balance between stretch, inflammation, ischemia, and biochemical mediators. Type I collagen mainly provides rigidity to the heart, whereas type III collagen contributes to elasticity. It has been shown that an increased collagen type I/III ratio, because of differential increase in collagens I and III, is usually observed in both dilated 5,6 and post–myocardial infarction cardiomyopathies, 7 resulting in systolic and diastolic dysfunctions. Moreover, it has been suggested that collagen I accumulation in heart failure may have adverse electrophysiologic consequences. 8 We hypothesized that measurement of biochemical markers indicative of specific pathways in cardiac collagen turnover might be more closely related to the occurrence of ventricular tachyarrhythmias. In this way, we could have valuable information regarding the possible identification of arrhythmogenic biochemical pathways in cardiac collagen turnover.
American Heart Journal Volume 164, Number 4
In this study, we prospectively investigated the relationship between life-threatening ventricular arrhythmias and cardiac collagen turnover indexes indicative of specific derangements in collagen physiology, such as (1) markers of collagen types I and III synthesis and their ratio (collagen I/III synthesis) and (2) markers of collagen degradation, degradation inhibition, and their ratio (equilibrium in myocardial collagen deposition/degradation). 9 Clinically stable patients with heart failure and a recently implanted ICD were assessed. Because inflammation has also been involved in the pathogenesis of sudden death, 10 we also assessed a marker of inflammation (interleukin-6 [IL-6]) for better understanding the mechanisms leading to the occurrence of ventricular arrhythmias. Left ventricular ejection fraction and N-terminal brain natriuretic peptide (NT-proBNP) were also systematically measured for the same reason.
Methods Patient selection The initially assessed study population consisted of 84 patients with clinically stable heart failure, due either to idiopathic dilated cardiomyopathy (n = 39) or coronary artery disease (n = 45) and an ICD. Patients with angina or signs of significant ischemia, as assessed by thallium scintigraphy or coronary angiography, were not included. Conditions known to alter collagen turnover or inflammatory status, such as renal impairment, liver disease, history of atrial fibrillation, pulmonary fibrosis, extensive wounds, metabolic bone disease, connective tissue disorders, malignancy, chronic inflammatory disease, corticosteroid treatment, and recent infection or surgery, were all considered as exclusion criteria. Patients were receiving a combination of aldosterone antagonists, angiotensin-converting enzyme inhibitors, or angiotensin II receptor blockers, β-blockers, and diuretics at a stable dose for at least 3 months before being included in the study. They were under optimized therapy for heart failure. All had an implanted ICD for secondary (n = 36) or primary prevention of sudden death according to current guidelines. 3 Before study initiation, none of the secondary prevention group patients had experienced aborted sudden death or a rapid ventricular tachycardia (N170 beat/min), which was our criterion for a potentially life-threatening arrhythmic event during study follow-up. Episodes during which the observed fast ventricular arrhythmia was thought to be due to a new episode of unstable angina or to recently deteriorated heart failure were not included in the analysis.
Study protocol We assessed markers of collagen type I synthesis (procollagen type I carboxyterminal peptide [PICP]), collagen type III synthesis (procollagen type III aminoterminal peptide [PIIINP]), and their ratio (PICP/PIIINP). Markers of collagen degradation (matrix metalloproteinase 9 [MMP-9]), collagen degradation inhibition (tissue inhibitor of MMP 1 [TIMP-1]), and their ratio were also assessed. Finally, IL-6 as well NT-proBNP levels was measured. Blood samples were obtained at the beginning of the study, before ICD implantation, by direct puncture of an
Flevari et al 531
antecubital vein after 20 minutes of supine rest. At the same 48-hour period, we assessed ejection fraction. 11
Follow-up After implantation and evaluation of device function, tachycardia detection rates and therapies were programmed. A ventricular tachycardia zone at 170 beat/min was programmed in all patients (initial antitachycardia pacing followed by shocks, if necessary). Above 200 beat/min, only shock therapies were programmed. All patients were prospectively followed up in the implantable device clinic of our department every 4 months or sooner in case of device discharges, thus ensuring comprehensive data collection. Data concerning arrhythmias and therapy were obtained and stored at the time of device interrogation on each follow-up visit. The incidence and type of arrhythmias and the incidence of appropriate and inappropriate defibrillator therapies were determined by reviewing stored electrograms. Events were classified by an agreement of 2 reviewing electrophysiologists. In case of an arrhythmic event with shock therapy, an appropriate laboratory evaluation, including cardiac troponin, plasma electrolytes, and thyroid hormones, to exclude potentially arrhythmia-triggering factors was carried out. If this was proved to be true, the specific episodes were not taken into account. At the end of a 12-month period, the total number of appropriate therapeutic interventions for ventricular tachyarrhythmias faster than 170 beat/min was assessed. In case of N1 intervention for the same arrhythmia, only the first one was taken into account. The number of these interventions was related to the assessed biochemical parameters.
Laboratory measurements For all parameters analyzed, blood samples were drawn in BD Vacutainer SST gel clotter tubes (BD Hellas, Athens, Greece) and were centrifuged within 30 minutes. The recovered sera were stored at −30°C until the analysis day. All samples were assayed in duplicate. We used commercially available enzyme-linked immunosorbent assay kits to measure serum concentrations of PICP and PIIINP (USCN Life Science, Wuhan, China) and IL-6, MMP-9, and TIMP-1 (Bender MedSystems, Vienna, Austria). All of the aforementioned kits used the following sandwich enzymelinked immunosorbent assay format: 96-well microtitre plates were already precoated with a murine monoclonal antibody against the substance being measured. Standards of the analyte, control samples, and patient samples were added in the wells and incubated at room temperature for 2 hours along with another biotinylated antibody directed against another epitope of the analyte. Then, after washing, streptavidin labeled with the horse radish peroxidase enzyme was added. After washing, a substrate for horse radish peroxidase, the chromogen tetra-methyl-benzidine was added and incubated for 30 minutes in the dark. The reaction was stopped with the addition of 2N H2SO4, and then the optical densities were read at 450 nm in the Elx microplate reader (Bio-tek Instruments, Highland Park, VE), and standard curves were plotted with the included KC4 software (Winooski, VT). Assays were valid when the control samples were measured within a specified range of the analyte concentration. All Bender MedSystems kits were CE-IVD marked. One of them,
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Table I. Characteristics of study patients (N = 74) Age (y) Male/female, n Heart failure etiology, n (%) Coronary artery disease Dilated cardiomyopathy Left ventricular ejection fraction (%) Left ventricular end-diastolic diameter (mm) NYHA, n (%) I II NT-proBNP (ng/L) Hemoglobin level (g/dL) Diabetes, n (%) Arterial hypertension, n (%) Medication, n (%) ACE inhibitors or ATII blockers β-blockers Amiodarone Statin Aldosterone antagonists
Table II. Serum markers of cardiac extracellular matrix turnover 56 ± 2 51/23 39 (53) 35 (47) 29 ± 1 61 ± 0.6 22 52 865 ± 4.8 14 ± 0.2 11 (15) 26 (35) 70 (95) 72 (97) 14 (19) 53 (72) 41 (55)
NYHA, New York Heart Association; ACE, angiotensin-converting enzyme; ATII, angiotensin II.
the IL-6 kit, was traceable to the international standard NIBSC 89/548; its measuring range was 1.56 to 100 pg/mL, and its limit of detection, 0.92 pg/mL. Other kits' limit of detection were 1.56 ng/mL for PICP, 15.6 pg/mL for PIIINP, 0.05 ng/mL for MMP-9, and, finally, 10 pg/mL for TIMP-1. Measurements were performed in duplicate by personnel blinded to the patients' clinical details. The intra- and interassay coefficient of variation were b8% and b10%, respectively, for all assays.
Statistical analysis Descriptive data are presented as mean ± SE for continuous variables and counts (proportions) for categorical variables. Simple and multiple stepwise regressions were used for analysis to explore the potential relation of the assessed biochemical markers with ensuing episodes. P b .01 was considered statistically significant for univariate analysis. For multivariate analysis, the value of significance was set to .05. The software Statistica (version 6.0) (Statsoft, Tulsa, OK) was used. The ethics committee of our institution approved the study. This investigation conforms to the principles outlined in the Declaration of Helsinki. Informed consent was obtained from all subjects before their participation in the study. No extramural funding was used to support this work. The authors are solely responsible for the design and conduct of this study, all study analyses, and drafting and editing of the manuscript.
Results
PICP (ng/mL) PIIINP (ng/mL) PICP/PIIINP MMP-9 (ng/mL) TIMP-1 (ng/mL) MMP-9/TIMP-1
3.3 44.2 65.5 778 635 405
± ± ± ± ± ±
0.86 2.6 14 70 46 193
Table III. Multiple regression summary results Variable MMP-9/TIMP-1 PICP/PIIINP Ejection fraction
Step +in/−out
Multiple R
F—to enter/ remove
P
1 2 3
0.33 0.45 0.51
7.79 7.71 4.64
.0068 .0072 .0350
patients were eventually analyzed. No patient died or withdrew during the study period. Their baseline characteristics are depicted in Table I.
Arrhythmic episodes and collagen turnover markers During the follow-up period, appropriate device therapies were delivered in 21 of the 74 patients. In total, 27 fast ventricular arrhythmic episodes were included in the analysis, as prespecified, with antitachycardia pacing in 5, antitachycardia pacing with shocks in 13, and shocks in 9 (2 of which were due to polymorphic arrhythmias faster than 200 beat/ min) . Among the 21 patients experiencing the episodes, 16 had 1 tachyarrhythmic event, and the remaining had ≥5 episodes. Two patients experienced inappropariate ICD discharge. Two arrhythmic episodes due to hypokalemia were not taken into account. Serum levels of cardiac extracellular matrix turnover markers are shown in Table II. After univariate analysis, significant relations were observed between the number of episodes and MMP-9/TIMP-1 (r = 0.33, P = .007), PICP/PIIINP (r = 0.32, P = .008), and ejection fraction (r = −0.32, P = .009). The relations remained significant after multivariate analysis regarding the 3 indexes (Table III, Figure 1). The number of arrhythmic episodes was not related to IL-6, although the P value almost reached significance (r = 0.25, P = .04). NT-proBNP was not related to the number of episodes (r = 0.21, P = .09). The number of episodes was not related to PICP (r = 0.15, P = .22), PIIINP (r = −0.013, P = .91), MMP-9 (r = 0.03, P = .79), or TIMP-1 (r = 0.15, P = .21). Among the assessed indexes, IL-6 showed a significant relation to ejection fraction (r = −0.32, P = .006) (Figure 2). Procollagen type I carboxyterminal peptide was related to PIIINP (r = 0.45, P = .0001) (Figure 3). No other significant relations were observed.
Patient characteristics During the 1-year follow-up, fast ventricular arrhythmias related to unstable angina or to heart failure deterioration were observed in 4 patients with dilated cardiomyopathy and in 6 with coronary artery disease; thus, these 10 patients were not included in the analysis. The data of the remaining 74
Discussion Main findings In this study, we have shown that, in stable patients with heart failure and an ICD, the biochemical markers
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Figure 1
Mean values of MMP-9/TIMP-1, PICP/PIIINP, left ventricular ejection fraction, and IL-6 in the groups of study patients with and without arrhythmias during follow-up. Significant differences were observed in these parameters between the 2 patient groups.
indicative of specific derangements in the balance of collagens I and III synthesis as well as in the equilibrium of myocardial deposition and degradation are related to the frequency of ensuing malignant ventricular arrhythmias, occurring in the 12-month period after ICD implantation. These indexes seem to be related to arrhythmic episodes as strongly as ejection fraction.
Fibrosis and ventricular arrhythmias Several preclinical and clinical studies suggest that myocardial fibrosis promotes ventricular arrhythmias. 12,13 Fibrosis affects conduction, especially in the transverse direction, the refractory period, and may result in conduction block, factors that are in favor of reentrant arrhythmias. 13 The uncoupling imposed by increased
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Figure 1 (continued )
collagen deposition is also in favor of the occurrence of arrhythmias by triggered activity 14 and enhanced automaticity. 15 Recent clinical data not only provide evidence for a mechanistic link between fibrosis and malignant ventricular arrhythmias but also suggest that absence of fibrosis predicts a low risk of implanted defibrillator therapy. 16
Markers of myocardial collagen degradation and arrhythmogenicity Biochemical markers of collagen turnover correlate significantly with fibrosis in endomyocardial biopsies 17 as well as in cardiac magnetic resonance imaging findings. 18-20 Such markers have been associated with
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Figure 2
Relation between left ventricular ejection fraction and IL-6.
the risk of ventricular tachycardia in certain patient subsets with coronary artery disease, having received an ICD for secondary prevention of SCD. 8 An interesting finding of this study has been that an increased ratio between MMP-9 and TIMP-1 levels showed the strongest association with ventricular arrhythmogenicity. Matrix metalloproteinase 9 is one of the MMPs, enzymes that are involved in degradation of extracellular matrix components. 21 Matrix metalloproteinase 9, in particular, has been shown to have a role in myosin heavy chain degradation, thus potentially contributing to systolic dysfunction. 22 Most of MMPs, including MMP-9, can be inhibited by a member of the family of tissue inhibitors of metalloproteinases, such as TIMP-1. The ratio between serum MMP/TIMP-1 concentration has been shown to be elevated in patients with heart failure 22 and has been related to a predominance of collagenolytic activity, playing an important role in heart failure remodeling. 22 This ratio has recently been proposed as a more accurate marker of MMP activity. 23 Because the ratio of circulating MMP/TIMP mirrors the equilibrium of collagen degradation within the myocardium and its inhibition, 16 it seems that this ratio is physiologically more important than the absolute values of MMP-9 and TIMP regarding the occurrence of malignant ventricular arrhythmias. This is physiologically in keeping with the present study results.
Markers of collagens I to III synthesis and ventricular arrhythmias The pathologic extracellular matrix turnover can be measured in the serum of patients with heart failure. 8 Significant results have been found regarding the ratio between markers of collagens I and III synthesis and ventricular arrhythmogenicity. Procollagen type I carboxyterminal peptide is a marker of the more rigid collagen I synthesis, whereas PIIINP a marker of the more elastic collagen III synthesis. 9 A delicate balance between synthesis and degradation of these 2 collagen types provides the physical support that maintains myocardial structure and heart electrophysiologic function. Although none of the 2 markers was directly related to the frequency of malignant arrhythmias, it was interesting to find that their ratio, expressing the impaired balance between rigid and elastic collagen synthesis, 5-7 was more important regarding future arrhythmogenesis than any one of the 2 markers of collagens I and III synthesis. Indeed, despite that PICP and PIIINP were weakly related (Figure 3), implying that they have the tendency to increase together, it seems that the disruption in extracellular matrix turnover in favor of collagen I may be the key arrhythmogenic element. Previous studies Two previous studies have similarly investigated the relationship between cardiac fibrosis and the incidence
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Figure 3
Relation between PIIINP and PICP.
of ventricular arrhythmias in patients with an ICD. The first, by Blangy et al, 8 examined secondary prevention patients with coronary artery disease; in accordance with our findings, they have shown that the combination of decreased procollagen type III and increased procollagen type I was a significant marker of the risk of ventricular tachycardia. A marker of inflammation, hs-CRP, was also related to ventricular arrhythmia occurrence, whereas the ratio between collagen degradation and degradation inhibition was not studied. An apparently contradicting result has been observed in our study regarding our finding that IL-6 was not strongly related to the number of arrhythmic episodes. This difference may be related to the different patient population and to the fact that we have assessed a different inflammation marker, IL-6. It is possible that—despite their similarities—hs-CRP and IL-6 reflect different aspects or stages in the inflammation process. However, they have both been previously associated with the occurrence of SCD. 10,24 The second study by Kanoupakis et al, 25 conducted in patients with idiopathic dilated cardiomyopathy and an ICD implanted prophylactically, has demonstrated a potential role for biochemical markers of collagen I turnover in risk assessment. However, they have not assessed collagen type III indexes, the ratio between collagen degradation and its inhibition, or inflammation markers. It is noteworthy that we have similar results regarding not only patients with dilated cardiomyopathy but also patients with coronary artery disease; this is in
accordance with the current concept that fibrosis that is remote from the infarct is a more significant determinant of adverse structural (and, possibly, electrical) remodeling in a similar way to that observed in dilated cardiomyopathy. 26
Implications of the study The new information regarding the balance in the types of collagen synthesis, the equilibrium in myocardial degradation and its inhibition, and their relation to ventricular tachyarrhythmias is compatible with approaches that encompass a more general and pathophysiologic evaluation of “vulnerability” to SCD, including markers indicative of multiple pathophysiologic mechanisms in the “multihit hypothesis of SCD pathogenesis. 27 Because it seems that specific pathways in extracellular matrix remodeling may be more “arrhythmia related” than others, further studies are warranted in search of combinations of more specific risk markers. Another potential application of our findings is that specific fibrosis biomarkers such as those assessed in this study may prove clinically useful toward a more accurate assessment of arrhythmic risk in patients with heart disease.
References 1. Vest III RN, Gold MR. Risk stratification of ventricular arrhythmias in patients with systolic heart failure. Curr Opin Cardiol 2010 (e-pub ahead of print).
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2. Tung R, Zimetbaum P, Josephson ME. A critical appraisal of implantable cardioverter-defibrillator therapy for the prevention of sudden cardiac death. J Am Coll Cardiol 2008;52:111-21. 3. Epstein AE, DiMarco JP, Ellenbogen KA, et al. ACC/AHA/HRS 2008 guidelines for device-based therapy of cardiac rhythm abnormalities: a report of the American College of Cardiology/ American Heart Association Task Force on Practice Guidelines (writing committee to revise the ACC/AHA/NASPE 2002 guideline update for implantation of cardiac pacemakers and antiarrhythmia devices). J Am Coll Cardiol 2008;51:e1-e62. 4. Buxton AE, Ellison KE, Lorvidhaya P, et al. Left ventricular ejection fraction for sudden death risk stratification and guiding implantable cardioverter-defibrillator implantation. J Cardiovasc Electrophysiol 2010;55:450-5. 5. Marijianowski MM, Teeling P, Mann J, et al. Dilated cardiomyopathy is associated with an increase in the type I/type III collagen ratio: a quantitative assessment. J Am Coll Cardiol 1995;25: 1263-72. 6. Pauschinger M, Knopf D, Petschauer S, et al. Dilated cardiomyopathy is associated with significant changes in type I/III ratio. Circulation 1999;99:2750-6. 7. Wei S, Chow LTC, Shum IOL, et al. Left and right ventricular collagen type I/III ratios and remodeling post-myocardial infarction. J Card Fail 1999;5:117-26. 8. Blangy H, Sadoul N, Dousset B, et al. Serum BNP, hs-C-reactive protein, procollagen to assess the risk of ventricular tachycardia in ICD recipients after myocardial infarction. Europace 2007;9: 724-9. 9. Klappacher G, Franzen P, Haab D, et al. Measuring extracellular matrix turnover in the serum of patients with idiopathic or ischemic dilated cardiomyopathy and impact on diagnosis and prognosis. Am J Cardiol 1995;75:913-8. 10. Ridker PM, Hennekens CH, Buring JE, et al. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med 2000;342:836-43. 11. Lang RM, Bierig M, Devereux RB, et al. Recommendations for chamber quatification: a report from the American Society of Echocardiography's Guidelines and Standards Committee and the Chamber Quantification Writing Group, developed in conjunction with the European Association of Echocardiography, a branch of the European Society of Cardiology. J Am Soc Echocardiogr 2005;18: 1440-63. 12. Spach MS, Boineau JP. Microfibrosis produces electrical load variations due to loss of side-to-side cell connections: a major mechanism of structural heart disease arrhythmias. Pacing Clin Electrophysiol 1997;20:397-413.
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13. de Bakker JM, van Capelle FJ, Janse MJ, et al. Slow conduction in the infarcted human heart. ‘Zigzag’ course of activation. Circulation 1993;88:915-26. 14. Morita N, Sovari AA, Xie Y, et al. Increased susceptibility of aged hearts to ventricular fibrillation during oxidative stress. Am J Physiol Heart Circ Physiol 2009;297:H1594-605. 15. Miragoli M, Sakvarini N, Rohr S. Myofibroblasts induce ectopic activity in cardiac tissue. Circ Res 2007;101:755-8. 16. Iles L, Pfluger H, Lefkovits L, et al. Myocardial fibrosis predicts appropriate device therapy in patients with implantable cardioverterdefibrillators for primary prevention of sudden cardiac death. J Am Coll Cardiol 2011;57:821-8. 17. Lopes B, Gonzalez A, Varo N, et al. Biochemical assessment of myocardial fibrosis in hypertensive heart disease. Hypertension 2001;38:1222-6. 18. Sipola P, Peuhkurinen K, Lauerma K, et al. Myocardial late gadolinium enhancement in associated with raised serum aminoterminal propeptide of type III collagen concentrations in patients with hypertrophic cardiomyopathy attributable to the Asp 175Asn mutation in the α tropomyosin gene: magnetic resonance imaging study. Heart 2006;92:1321-2. 19. Roldán V, Marín F, Gimeno JR, et al. Matrix metalloproteinases and tissue remodeling in hypertrophoc cardiomyopathy. Am Heart J 2008;156:85-91. 20. Orn S, Manhenke C, Squire IB, et al. Plasma MMP-2, MMP-9 and NBNP in long-term survivors following complicated myocardial infarction: relation to cardiac magnetic resonance imaging measures of left ventricular structure and function. J Card Fail 2007;13:843-9. 21. Cleutjens JP. The role of matrix metalloproteinases in heart disease. Cardiovasc Res 1996;32:816-21. 22. 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 Failure 2002;4:439-44. 23. De Jong S, van Veen TAB, de Bakker JMT, et al. Biomarkers of myocardial fibrosis. J Cardiovasc Pharmacol 2011;57:522-35. 24. Streither F, Kuschyk J, Veltmann C, et al. Role of proinflammatory markers and Nt-pro-BNP in patients with an implantable cardioverter-defibrillator and an electrical storm. Cytokine 2009;47:166-72. 25. Kanoupakis EM, Manios EG, Kallergis EM, et al. Serum markers of collagen turnover predict future shocks in implantable cardioverterdefibrillator recipients with dilated cardiomyopathy on optimal treatment. J Am Coll Cardiol 2010;55:2753-9. 26. Jellis C, Martin J, Narula J, et al. Assessment of nonischemic myocardial fibrosis. J Am Coll Cardiol 2010;56:89-97. 27. Tomaselli GF, Zipes D. What causes sudden death in heart failure? Circ Res 2004;95:754-63.
Serum markers of deranged myocardial collagen turnover: Their relation to malignant ventricular arrhythmias in cardioverter-defibrillator recipients with heart failure Panayota Flevari, MD, a George Theodorakis, MD, b Dionyssios Leftheriotis, MD, a Christos Kroupis, MD, c Fotis Kolokathis, MD, a Kleanthi Dima, MD, c Maria Anastasiou-Nana, MD, a and Dimitrios Kremastinos, MD a Athens, Greece
Background Pathologic collagen remodeling has been involved in the occurrence of ventricular arrhythmias and sudden cardiac death in heart failure. The aim of the study was to investigate the relationship between malignant ventricular arrhythmias and cardiac collagen turnover indexes, expressing specific types of derangement in collagen physiology, in stable patients with an implantable cardioverter-defibrillator (ICD). Methods Seventy-four patients with an ICD and heart failure were studied. They had coronary artery disease (n = 42) or dilated cardiomyopathy, New York Heart Association classes I and II, and left ventricular ejection fraction 29% ± 1%. An ICD had been implanted for secondary (n = 36) or primary prevention of sudden cardiac death. We assessed (1) markers of collagen types I and III synthesis and their ratio: procollagen type I carboxyterminal peptide (PICP), procollagen type III aminoterminal peptide (PIIINP), and PICP/PIIINP; (2) markers of collagen degradation, degradation inhibition, and their ratio: matrix metalloproteinase 9 (MMP-9), tissue inhibitor of matrix metalloproteinase (TIMP) 1 (TIMP-1), and MMP-9/TIMP-1. Patients were prospectively followed up for 1 year. The number of episodes necessitating appropriate interventions for ventricular tachyarrhythmias (N170 beat/min) was related to the assessed parameters. Results
Multivariate analysis revealed a significant relation between the number of tachyarrhythmic episodes and MMP9/TIMP-1 (P = .007), PICP/PIIINP (P = .007), and ejection fraction (P = .04). No other significant relation was observed between arrhythmias and the remaining parameters.
Conclusion In heart failure, biochemical markers indicative of a deranged equilirium in myocardial collagen deposition/degradation and collagen I/III synthesis are related to ventricular arrhythmogenesis. Further studies are needed to investigate their predictive ability. (Am Heart J 2012;164:530-7.)
The prevention of sudden cardiac death (SCD) among patients with heart failure remains suboptimal. 1 The use of implantable cardioverter-defibrillators (ICDs) is practically the most efficacious therapy, although it is expensive and not without side effects. 2-4 Changes in cardiac extracellular matrix characterize the remodeling process in heart failure, leading to fibrosis formation, collagen alteration, and degradation. Collagen synthesis and degradation result from a From the a2nd Department of Cardiology, Attikon University Hospital, Athens, Greece, b Department of Cardiology, Henry Dunant Hospital, Athens, Greece, and cBiochemical Laboratory, Attikon University Hospital, Athens, Greece. Submitted March 2, 2012; accepted July 13, 2012. Reprint requests: Panayota Flevari, MD, 2nd Department of Cardiology, Attikon University Hospital, Rimini 1, 12402 Haidari, Athens, Greece. E-mail: [email protected] 0002-8703/$ - see front matter © 2012, Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/j.ahj.2012.07.006
balance between stretch, inflammation, ischemia, and biochemical mediators. Type I collagen mainly provides rigidity to the heart, whereas type III collagen contributes to elasticity. It has been shown that an increased collagen type I/III ratio, because of differential increase in collagens I and III, is usually observed in both dilated 5,6 and post–myocardial infarction cardiomyopathies, 7 resulting in systolic and diastolic dysfunctions. Moreover, it has been suggested that collagen I accumulation in heart failure may have adverse electrophysiologic consequences. 8 We hypothesized that measurement of biochemical markers indicative of specific pathways in cardiac collagen turnover might be more closely related to the occurrence of ventricular tachyarrhythmias. In this way, we could have valuable information regarding the possible identification of arrhythmogenic biochemical pathways in cardiac collagen turnover.
American Heart Journal Volume 164, Number 4
In this study, we prospectively investigated the relationship between life-threatening ventricular arrhythmias and cardiac collagen turnover indexes indicative of specific derangements in collagen physiology, such as (1) markers of collagen types I and III synthesis and their ratio (collagen I/III synthesis) and (2) markers of collagen degradation, degradation inhibition, and their ratio (equilibrium in myocardial collagen deposition/degradation). 9 Clinically stable patients with heart failure and a recently implanted ICD were assessed. Because inflammation has also been involved in the pathogenesis of sudden death, 10 we also assessed a marker of inflammation (interleukin-6 [IL-6]) for better understanding the mechanisms leading to the occurrence of ventricular arrhythmias. Left ventricular ejection fraction and N-terminal brain natriuretic peptide (NT-proBNP) were also systematically measured for the same reason.
Methods Patient selection The initially assessed study population consisted of 84 patients with clinically stable heart failure, due either to idiopathic dilated cardiomyopathy (n = 39) or coronary artery disease (n = 45) and an ICD. Patients with angina or signs of significant ischemia, as assessed by thallium scintigraphy or coronary angiography, were not included. Conditions known to alter collagen turnover or inflammatory status, such as renal impairment, liver disease, history of atrial fibrillation, pulmonary fibrosis, extensive wounds, metabolic bone disease, connective tissue disorders, malignancy, chronic inflammatory disease, corticosteroid treatment, and recent infection or surgery, were all considered as exclusion criteria. Patients were receiving a combination of aldosterone antagonists, angiotensin-converting enzyme inhibitors, or angiotensin II receptor blockers, β-blockers, and diuretics at a stable dose for at least 3 months before being included in the study. They were under optimized therapy for heart failure. All had an implanted ICD for secondary (n = 36) or primary prevention of sudden death according to current guidelines. 3 Before study initiation, none of the secondary prevention group patients had experienced aborted sudden death or a rapid ventricular tachycardia (N170 beat/min), which was our criterion for a potentially life-threatening arrhythmic event during study follow-up. Episodes during which the observed fast ventricular arrhythmia was thought to be due to a new episode of unstable angina or to recently deteriorated heart failure were not included in the analysis.
Study protocol We assessed markers of collagen type I synthesis (procollagen type I carboxyterminal peptide [PICP]), collagen type III synthesis (procollagen type III aminoterminal peptide [PIIINP]), and their ratio (PICP/PIIINP). Markers of collagen degradation (matrix metalloproteinase 9 [MMP-9]), collagen degradation inhibition (tissue inhibitor of MMP 1 [TIMP-1]), and their ratio were also assessed. Finally, IL-6 as well NT-proBNP levels was measured. Blood samples were obtained at the beginning of the study, before ICD implantation, by direct puncture of an
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antecubital vein after 20 minutes of supine rest. At the same 48-hour period, we assessed ejection fraction. 11
Follow-up After implantation and evaluation of device function, tachycardia detection rates and therapies were programmed. A ventricular tachycardia zone at 170 beat/min was programmed in all patients (initial antitachycardia pacing followed by shocks, if necessary). Above 200 beat/min, only shock therapies were programmed. All patients were prospectively followed up in the implantable device clinic of our department every 4 months or sooner in case of device discharges, thus ensuring comprehensive data collection. Data concerning arrhythmias and therapy were obtained and stored at the time of device interrogation on each follow-up visit. The incidence and type of arrhythmias and the incidence of appropriate and inappropriate defibrillator therapies were determined by reviewing stored electrograms. Events were classified by an agreement of 2 reviewing electrophysiologists. In case of an arrhythmic event with shock therapy, an appropriate laboratory evaluation, including cardiac troponin, plasma electrolytes, and thyroid hormones, to exclude potentially arrhythmia-triggering factors was carried out. If this was proved to be true, the specific episodes were not taken into account. At the end of a 12-month period, the total number of appropriate therapeutic interventions for ventricular tachyarrhythmias faster than 170 beat/min was assessed. In case of N1 intervention for the same arrhythmia, only the first one was taken into account. The number of these interventions was related to the assessed biochemical parameters.
Laboratory measurements For all parameters analyzed, blood samples were drawn in BD Vacutainer SST gel clotter tubes (BD Hellas, Athens, Greece) and were centrifuged within 30 minutes. The recovered sera were stored at −30°C until the analysis day. All samples were assayed in duplicate. We used commercially available enzyme-linked immunosorbent assay kits to measure serum concentrations of PICP and PIIINP (USCN Life Science, Wuhan, China) and IL-6, MMP-9, and TIMP-1 (Bender MedSystems, Vienna, Austria). All of the aforementioned kits used the following sandwich enzymelinked immunosorbent assay format: 96-well microtitre plates were already precoated with a murine monoclonal antibody against the substance being measured. Standards of the analyte, control samples, and patient samples were added in the wells and incubated at room temperature for 2 hours along with another biotinylated antibody directed against another epitope of the analyte. Then, after washing, streptavidin labeled with the horse radish peroxidase enzyme was added. After washing, a substrate for horse radish peroxidase, the chromogen tetra-methyl-benzidine was added and incubated for 30 minutes in the dark. The reaction was stopped with the addition of 2N H2SO4, and then the optical densities were read at 450 nm in the Elx microplate reader (Bio-tek Instruments, Highland Park, VE), and standard curves were plotted with the included KC4 software (Winooski, VT). Assays were valid when the control samples were measured within a specified range of the analyte concentration. All Bender MedSystems kits were CE-IVD marked. One of them,
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Table I. Characteristics of study patients (N = 74) Age (y) Male/female, n Heart failure etiology, n (%) Coronary artery disease Dilated cardiomyopathy Left ventricular ejection fraction (%) Left ventricular end-diastolic diameter (mm) NYHA, n (%) I II NT-proBNP (ng/L) Hemoglobin level (g/dL) Diabetes, n (%) Arterial hypertension, n (%) Medication, n (%) ACE inhibitors or ATII blockers β-blockers Amiodarone Statin Aldosterone antagonists
Table II. Serum markers of cardiac extracellular matrix turnover 56 ± 2 51/23 39 (53) 35 (47) 29 ± 1 61 ± 0.6 22 52 865 ± 4.8 14 ± 0.2 11 (15) 26 (35) 70 (95) 72 (97) 14 (19) 53 (72) 41 (55)
NYHA, New York Heart Association; ACE, angiotensin-converting enzyme; ATII, angiotensin II.
the IL-6 kit, was traceable to the international standard NIBSC 89/548; its measuring range was 1.56 to 100 pg/mL, and its limit of detection, 0.92 pg/mL. Other kits' limit of detection were 1.56 ng/mL for PICP, 15.6 pg/mL for PIIINP, 0.05 ng/mL for MMP-9, and, finally, 10 pg/mL for TIMP-1. Measurements were performed in duplicate by personnel blinded to the patients' clinical details. The intra- and interassay coefficient of variation were b8% and b10%, respectively, for all assays.
Statistical analysis Descriptive data are presented as mean ± SE for continuous variables and counts (proportions) for categorical variables. Simple and multiple stepwise regressions were used for analysis to explore the potential relation of the assessed biochemical markers with ensuing episodes. P b .01 was considered statistically significant for univariate analysis. For multivariate analysis, the value of significance was set to .05. The software Statistica (version 6.0) (Statsoft, Tulsa, OK) was used. The ethics committee of our institution approved the study. This investigation conforms to the principles outlined in the Declaration of Helsinki. Informed consent was obtained from all subjects before their participation in the study. No extramural funding was used to support this work. The authors are solely responsible for the design and conduct of this study, all study analyses, and drafting and editing of the manuscript.
Results
PICP (ng/mL) PIIINP (ng/mL) PICP/PIIINP MMP-9 (ng/mL) TIMP-1 (ng/mL) MMP-9/TIMP-1
3.3 44.2 65.5 778 635 405
± ± ± ± ± ±
0.86 2.6 14 70 46 193
Table III. Multiple regression summary results Variable MMP-9/TIMP-1 PICP/PIIINP Ejection fraction
Step +in/−out
Multiple R
F—to enter/ remove
P
1 2 3
0.33 0.45 0.51
7.79 7.71 4.64
.0068 .0072 .0350
patients were eventually analyzed. No patient died or withdrew during the study period. Their baseline characteristics are depicted in Table I.
Arrhythmic episodes and collagen turnover markers During the follow-up period, appropriate device therapies were delivered in 21 of the 74 patients. In total, 27 fast ventricular arrhythmic episodes were included in the analysis, as prespecified, with antitachycardia pacing in 5, antitachycardia pacing with shocks in 13, and shocks in 9 (2 of which were due to polymorphic arrhythmias faster than 200 beat/ min) . Among the 21 patients experiencing the episodes, 16 had 1 tachyarrhythmic event, and the remaining had ≥5 episodes. Two patients experienced inappropariate ICD discharge. Two arrhythmic episodes due to hypokalemia were not taken into account. Serum levels of cardiac extracellular matrix turnover markers are shown in Table II. After univariate analysis, significant relations were observed between the number of episodes and MMP-9/TIMP-1 (r = 0.33, P = .007), PICP/PIIINP (r = 0.32, P = .008), and ejection fraction (r = −0.32, P = .009). The relations remained significant after multivariate analysis regarding the 3 indexes (Table III, Figure 1). The number of arrhythmic episodes was not related to IL-6, although the P value almost reached significance (r = 0.25, P = .04). NT-proBNP was not related to the number of episodes (r = 0.21, P = .09). The number of episodes was not related to PICP (r = 0.15, P = .22), PIIINP (r = −0.013, P = .91), MMP-9 (r = 0.03, P = .79), or TIMP-1 (r = 0.15, P = .21). Among the assessed indexes, IL-6 showed a significant relation to ejection fraction (r = −0.32, P = .006) (Figure 2). Procollagen type I carboxyterminal peptide was related to PIIINP (r = 0.45, P = .0001) (Figure 3). No other significant relations were observed.
Patient characteristics During the 1-year follow-up, fast ventricular arrhythmias related to unstable angina or to heart failure deterioration were observed in 4 patients with dilated cardiomyopathy and in 6 with coronary artery disease; thus, these 10 patients were not included in the analysis. The data of the remaining 74
Discussion Main findings In this study, we have shown that, in stable patients with heart failure and an ICD, the biochemical markers
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Figure 1
Mean values of MMP-9/TIMP-1, PICP/PIIINP, left ventricular ejection fraction, and IL-6 in the groups of study patients with and without arrhythmias during follow-up. Significant differences were observed in these parameters between the 2 patient groups.
indicative of specific derangements in the balance of collagens I and III synthesis as well as in the equilibrium of myocardial deposition and degradation are related to the frequency of ensuing malignant ventricular arrhythmias, occurring in the 12-month period after ICD implantation. These indexes seem to be related to arrhythmic episodes as strongly as ejection fraction.
Fibrosis and ventricular arrhythmias Several preclinical and clinical studies suggest that myocardial fibrosis promotes ventricular arrhythmias. 12,13 Fibrosis affects conduction, especially in the transverse direction, the refractory period, and may result in conduction block, factors that are in favor of reentrant arrhythmias. 13 The uncoupling imposed by increased
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Figure 1 (continued )
collagen deposition is also in favor of the occurrence of arrhythmias by triggered activity 14 and enhanced automaticity. 15 Recent clinical data not only provide evidence for a mechanistic link between fibrosis and malignant ventricular arrhythmias but also suggest that absence of fibrosis predicts a low risk of implanted defibrillator therapy. 16
Markers of myocardial collagen degradation and arrhythmogenicity Biochemical markers of collagen turnover correlate significantly with fibrosis in endomyocardial biopsies 17 as well as in cardiac magnetic resonance imaging findings. 18-20 Such markers have been associated with
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Figure 2
Relation between left ventricular ejection fraction and IL-6.
the risk of ventricular tachycardia in certain patient subsets with coronary artery disease, having received an ICD for secondary prevention of SCD. 8 An interesting finding of this study has been that an increased ratio between MMP-9 and TIMP-1 levels showed the strongest association with ventricular arrhythmogenicity. Matrix metalloproteinase 9 is one of the MMPs, enzymes that are involved in degradation of extracellular matrix components. 21 Matrix metalloproteinase 9, in particular, has been shown to have a role in myosin heavy chain degradation, thus potentially contributing to systolic dysfunction. 22 Most of MMPs, including MMP-9, can be inhibited by a member of the family of tissue inhibitors of metalloproteinases, such as TIMP-1. The ratio between serum MMP/TIMP-1 concentration has been shown to be elevated in patients with heart failure 22 and has been related to a predominance of collagenolytic activity, playing an important role in heart failure remodeling. 22 This ratio has recently been proposed as a more accurate marker of MMP activity. 23 Because the ratio of circulating MMP/TIMP mirrors the equilibrium of collagen degradation within the myocardium and its inhibition, 16 it seems that this ratio is physiologically more important than the absolute values of MMP-9 and TIMP regarding the occurrence of malignant ventricular arrhythmias. This is physiologically in keeping with the present study results.
Markers of collagens I to III synthesis and ventricular arrhythmias The pathologic extracellular matrix turnover can be measured in the serum of patients with heart failure. 8 Significant results have been found regarding the ratio between markers of collagens I and III synthesis and ventricular arrhythmogenicity. Procollagen type I carboxyterminal peptide is a marker of the more rigid collagen I synthesis, whereas PIIINP a marker of the more elastic collagen III synthesis. 9 A delicate balance between synthesis and degradation of these 2 collagen types provides the physical support that maintains myocardial structure and heart electrophysiologic function. Although none of the 2 markers was directly related to the frequency of malignant arrhythmias, it was interesting to find that their ratio, expressing the impaired balance between rigid and elastic collagen synthesis, 5-7 was more important regarding future arrhythmogenesis than any one of the 2 markers of collagens I and III synthesis. Indeed, despite that PICP and PIIINP were weakly related (Figure 3), implying that they have the tendency to increase together, it seems that the disruption in extracellular matrix turnover in favor of collagen I may be the key arrhythmogenic element. Previous studies Two previous studies have similarly investigated the relationship between cardiac fibrosis and the incidence
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Figure 3
Relation between PIIINP and PICP.
of ventricular arrhythmias in patients with an ICD. The first, by Blangy et al, 8 examined secondary prevention patients with coronary artery disease; in accordance with our findings, they have shown that the combination of decreased procollagen type III and increased procollagen type I was a significant marker of the risk of ventricular tachycardia. A marker of inflammation, hs-CRP, was also related to ventricular arrhythmia occurrence, whereas the ratio between collagen degradation and degradation inhibition was not studied. An apparently contradicting result has been observed in our study regarding our finding that IL-6 was not strongly related to the number of arrhythmic episodes. This difference may be related to the different patient population and to the fact that we have assessed a different inflammation marker, IL-6. It is possible that—despite their similarities—hs-CRP and IL-6 reflect different aspects or stages in the inflammation process. However, they have both been previously associated with the occurrence of SCD. 10,24 The second study by Kanoupakis et al, 25 conducted in patients with idiopathic dilated cardiomyopathy and an ICD implanted prophylactically, has demonstrated a potential role for biochemical markers of collagen I turnover in risk assessment. However, they have not assessed collagen type III indexes, the ratio between collagen degradation and its inhibition, or inflammation markers. It is noteworthy that we have similar results regarding not only patients with dilated cardiomyopathy but also patients with coronary artery disease; this is in
accordance with the current concept that fibrosis that is remote from the infarct is a more significant determinant of adverse structural (and, possibly, electrical) remodeling in a similar way to that observed in dilated cardiomyopathy. 26
Implications of the study The new information regarding the balance in the types of collagen synthesis, the equilibrium in myocardial degradation and its inhibition, and their relation to ventricular tachyarrhythmias is compatible with approaches that encompass a more general and pathophysiologic evaluation of “vulnerability” to SCD, including markers indicative of multiple pathophysiologic mechanisms in the “multihit hypothesis of SCD pathogenesis. 27 Because it seems that specific pathways in extracellular matrix remodeling may be more “arrhythmia related” than others, further studies are warranted in search of combinations of more specific risk markers. Another potential application of our findings is that specific fibrosis biomarkers such as those assessed in this study may prove clinically useful toward a more accurate assessment of arrhythmic risk in patients with heart disease.
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