Clin Res Cardiol DOI 10.1007/s00392-015-0871-y
ORIGINAL PAPER
Endomyocardial expression of SDF-1 predicts mortality in patients with suspected myocarditis Christine S. Zuern1 • Britta Walker2 • Martina Sauter3 • Malte Schaub1 • Madhumita Chatterjee1 • Karin Mueller1 • Dominik Rath1 • Sebastian Vogel1 Roland Tegtmeyer1 • Peter Seizer1 • Tobias Geisler1 • Reinhard Kandolf3 • Florian Lang2 • Karin Klingel3 • Meinrad Gawaz1 • Oliver Borst1,2
•
Received: 23 January 2015 / Accepted: 18 May 2015 Ó Springer-Verlag Berlin Heidelberg 2015
Abstract Background Risk stratification in patients with suspected myocarditis is pivotal for optimizing therapy. Stromal cellderived factor 1 (SDF-1) is an inflammatory chemokine expressed in the inflamed and failing myocardium. Therefore, we aimed to investigate whether endomyocardial expression of SDF-1 identifies high-risk patients with suspected myocarditis. Methods and results We prospectively enrolled 174 patients with non-ischemic HF who underwent endomyocardial biopsy for suspected myocarditis. Biopsies were analyzed using established histopathological and immunohistological criteria together with SDF-1 staining. SDF-1 was significantly enhanced in patients with inflammatory cardiomyopathy (65.4 % positive biopsies) as compared to patients with noninflammatory cardiomyopathy (19.1 %, p \ 0.001). SDF-1 expression levels correlated significantly with the degree of myocardial fibrosis (correlation coefficient r = 0.196; p = 0.010) since patients with severe myocardial fibrosis
displayed high myocardial SDF-1 expression. During a mean follow-up of 27.5 months, 20 patients (11.5 %) died. The 4-year mortality rate was 26.0 % among the 92 SDF-1-positive patients vs. 9.5 % among the 82 SDF-1-negative patients (p = 0.001). On multivariable analysis which considered clinical (NYHA functional class, left ventricular ejection fraction), laboratory (brain natriuretic peptide, troponin I) and biopsy staining, SDF-1 was the strongest independent predictor of mortality (hazard ratio 6.1; 95 % confidence interval 1.4–27.5; p = 0.018). Subgroup analysis revealed SDF-1 as a predictor of mortality in both patients with inflammatory and non-inflammatory cardiomyopathy. Conclusions Endomyocardial expression of SDF-1 is enhanced in inflammatory cardiomyopathy, positively correlates with myocardial fibrosis and identifies high-risk patients with suspected myocarditis.
Electronic supplementary material The online version of this article (doi:10.1007/s00392-015-0871-y) contains supplementary material, which is available to authorized users.
Introduction
& Christine S. Zuern [email protected] Meinrad Gawaz [email protected] 1
Department of Cardiology and Cardiovascular Medicine, University of Tuebingen, Otfried-Mueller-Str. 10, 72076 Tuebingen, Germany
2
Department of Physiology, University of Tuebingen, Gmelinstr. 5, 72076 Tuebingen, Germany
3
Department of Molecular Pathology, University of Tuebingen, Liebermeisterstr. 8, 72076 Tuebingen, Germany
Keywords Myocarditis Cardiomyopathy Biomarker SDF-1 Endomyocardial biopsy
Over the last decades, huge efforts have been made in both medical and device-based therapy of cardiomyopathies. However, the prognosis remains poor so far [1]. Myocarditis represents a significant subgroup which critically contributes to the global burden of chronic heart failure (HF) [2]. The clinical presentation of myocarditis is variable. Symptoms yield from mild dyspnoea or chest pain to arrhythmias, cardiogenic shock or terminal HF with need for left ventricular assist device and heart transplantation [2–5]. A variety of risk predictors have been identified in patients with suspected myocarditis and heart failure, such as advanced NYHA class [6], impaired left ventricular
123
Clin Res Cardiol
ejection fraction (LVEF) [7], cardiac biomarkers [8] such as brain natriuretic peptides (BNP) [9], troponins [10], soluble ST2 [11], Galectin-3 [12], high-mobility-group box 1 [13], heart rate at rest [14] or immunohistological signs of inflammation [15]. However, due to the diverse presentation of myocarditis and the widely varying individual course of progression of the disease, early and accurate diagnosis of myocarditis and identification of novel reliable risk factors that help to confirm the diagnosis of myocarditis and identify high-risk patients are still warranted. Chemokines interacting with their receptors play a crucial role in the recruitment of inflammatory cells to myocarditis lesions and are of central importance for initiation, maintenance and resolution of inflammation [16]. Recent studies demonstrated cardiodepressive effects of a number of chemokines including CX3CL1/fractalkine [16]. SDF-1, also known as CXCL12, is an inflammatory CXC-chemokine that is rapidly and transiently upregulated in response to tissue injury [17]. In the heart, SDF-1 is expressed by stromal cells, endothelial cells, fibroblasts, dendritic cells and cardiomyocytes [18, 19]. SDF-1 binds to its receptors CXCR4 and CXCR7 regulating adhesion and migration of inflammatory cells including platelets, monocytes and lymphocytes [20– 22]. SDF-1 further increases homing of circulating CXCR4positive progenitor cells to ischemic tissue [23], and plays a major role in cell proliferation and ventricular remodeling in ischemic cardiomyopathy [19, 24]. We and other groups were able to show that SDF-1 is cardioprotective after myocardial hypoxemia and increases recruitment of circulating progenitor cells promoting angiogenesis and supporting myocyte survival [22, 24, 25]. In recent studies, we observed an association between platelet SDF-1 expression and reduced left ventricular function due to ischemic cardiomyopathy [26], as well as the number of circulating CD34? progenitor cells and platelet-progenitor cells co-aggregates augmenting recruitment of CD34? cells into ischemic microcirculation [25, 27]. Additionally, it was recently shown that increased plasma SDF-1 levels were associated with development of HF and all-cause mortality [28]. However, the role of the inflammatory chemokine SDF-1, which is transiently expressed in the myocardium upon cardiomyocyte damage, in myocarditis is still unknown. We therefore explored whether endomyocardial expression of SDF-1 may serve as predictor of mortality in a cohort of patients undergoing endomyocardial biopsy for suspected myocarditis.
Materials and methods Patients We prospectively included 174 consecutive patients who underwent endomyocardial biopsy (EMB) between 3/2009
123
and 6/2011 as part of an evaluation for suspected myocarditis at our university center. Indications for EMB were based on the current guidelines [29]. Patients were included if they clinically presented with signs of possible myocarditis such as history of recent infection, impaired global or regional left ventricular function, increase in serum concentrations of cardiac biomarkers, pericardial effusion, sustained or non-sustained ventricular tachycardia of unknown origin. Patients with significant coronary artery disease as the underlying cause of HF were excluded. All patients received medication according to current ESC and ACC/AHA guidelines depending on their LVEF and HF symptoms [30]. The study was approved by the local ethics committee. Informed consent was obtained from all patients. Assessment of clinical risk factors NYHA functional class was assessed by experienced cardiologists at study entry. LVEF was estimated by echocardiography (iE33, Philips Medical Systems) by modified Simpson rule with images obtained from apical 4and 2-chamber views. Laboratory studies included BNP and troponin I (TnI). BNP and TnI levels were assessed by immunoassay (ADVIA Centaur BNP Assay and ADVIA Centaur TnI-Ultra, Siemens Healthcare Diagnostics, respectively). Patients were classified as BNP-positive or TnI-positive if BNP level was above 100 ng/L or TnI level above 0.04 lg/L, respectively. Analysis of endomyocardial biopsies Approximately five EMB specimens were taken with a dedicated bioptome (Biopsy Forceps, Cordis Corporation, USA) from the right ventricular septum. Three biopsy specimens were fixed in 4 % buffered formaldehyde for immunohistology. Two tissue samples were fixed in RNAlater (Ambion Inc., Foster City, USA) for detection of viral genomes by nested real time polymerase chain reaction (RTPCR). Paraffin-embedded EMB were stained with Masson’s trichrome reflecting myocyte necrosis, as well as interstitial fibrosis and analyzed by light microscopy [31, 32]. Histological analysis followed the Dallas criteria [32] complemented by immunohistology to assess inflammation. According to the Dallas criteria [32], inflammatory cardiomyopathy was defined by lymphocytic infiltrates in association with myocyte necrosis. According to the WHO/ International Society and Federation of Cardiology Task Force on the Definition and Classification of Cardiomyopathies [33], inflammation was considered present after immunohistological detection of mononuclear infiltrates with [14 leucocytes per 1 mm2 in the myocardium, in
Clin Res Cardiol
addition to enhanced expression of HLA class II molecules. EMB without signs of inflammation but with myocardial damage and fibrosis were classified as non-inflammatory cardiomyopathy. Those patients suffered predominantly from idiopathic dilative cardiomyopathy or hypertensive heart disease. According to established scores for evaluation of CD68 and MHCII [1, 15, 34, 35], sections stained for SDF-1 were analyzed by a semiquantitative score ranging from 1 (no expression) to 4 (ubiquitous high expression) by two coinvestigators in a blinded manner from one to two sections for each staining [36]. Fleiss statistics revealed a free marginal kappa of 0.7 for SDF-1 indicating a good agreement between the operators. EMB stained for SDF-1 were then classified in a dichotomized way as ‘‘negative’’ (no/ low) or ‘‘positive’’ (moderate/high expression). The extent of myocardial fibrosis in EMB was defined as an index as described previously [37]. The percentage area of fibrosis in the section was evaluated by dividing the sum of the fibrotic areas of the section by that of the total tissue area. According to this fibrosis index, patients were classified as having no/mild, moderate, or severe fibrosis. Follow-up and study endpoints 132 of the 174 patients presented in our outpatient clinics for follow-up visits scheduled at 6-months intervals. For the remaining 42 patients who were not seen in our clinics, telephone contact was made to determine occurrence of an endpoint. None of the patients were lost to follow-up. The primary study endpoint was death from all causes. The secondary endpoint was defined as the composite of the rate of death from all causes and HF-related rehospitalizations. Physicians who were in charge of decision for rehospitalizations were not involved in the study. Statistical analysis Continuous variables are expressed as median and interquartile range (IQR) and compared using Mann–Whitney U test. Categorical data are presented as proportions and analyzed by Chi square test. The following risk factors were prospectively assessed: NYHA functional class, LVEF, BNP, TnI and SDF-1. Continuous parameters were dichotomized at established cut-off values. Multivariable analyses were performed using a Cox proportional hazards model with the enter procedure, and adjusted to age and gender. Predefined subgroup analyses were performed for patients with inflammatory and non-inflammatory cardiomyopathy. Survival curves of patients grouped by prespecified variables were calculated by Kaplan–Meier analyses and compared with log-rank test. Time point for beginning of survival analysis was the date of EMB. As a
measure of agreement between the different blinded raters of EMB staining, Fleiss’ kappa coefficient was calculated [38]. A receiver-operator characteristic (ROC) curve for prediction of mortality was constructed for SDF-1 by plotting the dependency of specificity on sensitivity. The ROC curve was quantified by the integral of the curve [area under the curve (AUC)]. Confidence intervals (CIs) were estimated using bootstrapping. Correlations between SDF1 and fibrosis, as well as with CD3, MHCII and LVEF were tested with Spearman rank correlation. Comparisons were considered statistically significant if two-sided p value was \0.05. Statistical analyses were performed using SPSS software version 21 (SPSS Inc.).
Results Baseline characteristics of the study cohort are presented in Table 1. Patients were rather young [median age 53 (IQR 45–67) years] and predominantly males (76 %). 83 % of the patients had HF symptoms (NYHA functional class CII). LVEF was impaired with \40 % in 51 % of the patients. 75 and 29 % of patients presented with elevated serum concentrations of BNP and TnI, respectively. 73 % of the EMBs (n = 127) showed myocardial inflammation. 8/127 patients presented with histopathological and immunohistological signs of acute myocarditis and the remaining 119/127 patients with chronic myocarditis. 64 of the 174 patients (36.8 %) were virus-positive. From those patients, 34/174 (19.5 %) were parvovirus B19 (PVB19) positive, 19/174 patients (10.9 %) were Epstein Barr virus (EBV) positive, 17/174 patients (9.8 %) were human herpesvirus 6 (HHV6) positive and 1/174 (0.6 %) coxsackie B3 (CVB3) positive. Three patients had a co-infection of PVB19 and HHV6, three patients PVB19 and EBV, and one patient EBV as well as HHV6. 53 % of the patients had a SDF-1 positive EMB. SDF-1positive patients had a higher prevalence of myocardial inflammation (90 vs. 54 %, p \ 0.0001) with higher amounts of CD3? T cells (58 vs. 16 %, p \ 0.0001), CD68? macrophages (76 vs. 18 %, p \ 0.0001) and enhanced expression of MHCII (76 vs. 18 %, p \ 0.0001), when compared to SDF-1 negative patients (Fig. 1b). Furthermore, patients with SDF-1 positive EMB were significantly more often virus-positive (45 vs. 28 %, p = 0.024) and TnI-positive (37 vs. 20 %, p = 0.011). In EMB of patients with myocardial inflammation, SDF-1 was predominantly present in endothelial cells and interstitial cells, most likely representing fibroblasts and immune cells, e.g. macrophages and dendritic cells (Fig. 1a). SDF-1 was not expressed in the healthy human myocardium of control subjects (Fig. 1a). Of note, all patients with acute myocarditis (100 %) and 75/119 (63.0 %) of
123
Clin Res Cardiol Table 1 Baseline characteristics of the cohort of patients with suspected myocarditis All patients (n = 174)
Survivors (n = 154)
Non-survivors (n = 20)
p value
Demographics Age (years)
53 (45–67)
Males (%)
133 (76.4) 2
Body mass index (kg/m )
25 (23–29)
53 (44–66) 118 (76.6) 26 (24–29)
62 (49–71)
0.123
15 (75.0)
0.872
24 (22–29)
0.112
Risk variables NYHA class CII (%)
144 (82.8)
128 (83.1)
16 (80.0)
0.728
88 (50.6)
73 (47.4)
15 (75.0)
0.020
LVEF \40 % BNP-positivea (%)
131 (75.3)
112 (72.7)
19 (95.0)
0.030
Troponin I positivea (%)
50 (28.7)
40 (26.0)
10 (50.0)
0.025
PAPsys (mmHg)
24 (20–35)
24 (20–33)
32 (21–41)
0.047
Results of endomyocardial biopsy (%) Inflammatory CMP 127 (73.0)
112 (72.7)
15 (75.0)
0.829
Non-inflammatory CMP
47 (27.0)
42 (27.3)
5 (25.0)
0.829
Virus-positive
64 (36.8)
57 (37.0)
7 (35.0)
0.861
SDF-1-positive
92 (52.9)
74 (48.1)
18 (90.0)
ORIGINAL PAPER
Endomyocardial expression of SDF-1 predicts mortality in patients with suspected myocarditis Christine S. Zuern1 • Britta Walker2 • Martina Sauter3 • Malte Schaub1 • Madhumita Chatterjee1 • Karin Mueller1 • Dominik Rath1 • Sebastian Vogel1 Roland Tegtmeyer1 • Peter Seizer1 • Tobias Geisler1 • Reinhard Kandolf3 • Florian Lang2 • Karin Klingel3 • Meinrad Gawaz1 • Oliver Borst1,2
•
Received: 23 January 2015 / Accepted: 18 May 2015 Ó Springer-Verlag Berlin Heidelberg 2015
Abstract Background Risk stratification in patients with suspected myocarditis is pivotal for optimizing therapy. Stromal cellderived factor 1 (SDF-1) is an inflammatory chemokine expressed in the inflamed and failing myocardium. Therefore, we aimed to investigate whether endomyocardial expression of SDF-1 identifies high-risk patients with suspected myocarditis. Methods and results We prospectively enrolled 174 patients with non-ischemic HF who underwent endomyocardial biopsy for suspected myocarditis. Biopsies were analyzed using established histopathological and immunohistological criteria together with SDF-1 staining. SDF-1 was significantly enhanced in patients with inflammatory cardiomyopathy (65.4 % positive biopsies) as compared to patients with noninflammatory cardiomyopathy (19.1 %, p \ 0.001). SDF-1 expression levels correlated significantly with the degree of myocardial fibrosis (correlation coefficient r = 0.196; p = 0.010) since patients with severe myocardial fibrosis
displayed high myocardial SDF-1 expression. During a mean follow-up of 27.5 months, 20 patients (11.5 %) died. The 4-year mortality rate was 26.0 % among the 92 SDF-1-positive patients vs. 9.5 % among the 82 SDF-1-negative patients (p = 0.001). On multivariable analysis which considered clinical (NYHA functional class, left ventricular ejection fraction), laboratory (brain natriuretic peptide, troponin I) and biopsy staining, SDF-1 was the strongest independent predictor of mortality (hazard ratio 6.1; 95 % confidence interval 1.4–27.5; p = 0.018). Subgroup analysis revealed SDF-1 as a predictor of mortality in both patients with inflammatory and non-inflammatory cardiomyopathy. Conclusions Endomyocardial expression of SDF-1 is enhanced in inflammatory cardiomyopathy, positively correlates with myocardial fibrosis and identifies high-risk patients with suspected myocarditis.
Electronic supplementary material The online version of this article (doi:10.1007/s00392-015-0871-y) contains supplementary material, which is available to authorized users.
Introduction
& Christine S. Zuern [email protected] Meinrad Gawaz [email protected] 1
Department of Cardiology and Cardiovascular Medicine, University of Tuebingen, Otfried-Mueller-Str. 10, 72076 Tuebingen, Germany
2
Department of Physiology, University of Tuebingen, Gmelinstr. 5, 72076 Tuebingen, Germany
3
Department of Molecular Pathology, University of Tuebingen, Liebermeisterstr. 8, 72076 Tuebingen, Germany
Keywords Myocarditis Cardiomyopathy Biomarker SDF-1 Endomyocardial biopsy
Over the last decades, huge efforts have been made in both medical and device-based therapy of cardiomyopathies. However, the prognosis remains poor so far [1]. Myocarditis represents a significant subgroup which critically contributes to the global burden of chronic heart failure (HF) [2]. The clinical presentation of myocarditis is variable. Symptoms yield from mild dyspnoea or chest pain to arrhythmias, cardiogenic shock or terminal HF with need for left ventricular assist device and heart transplantation [2–5]. A variety of risk predictors have been identified in patients with suspected myocarditis and heart failure, such as advanced NYHA class [6], impaired left ventricular
123
Clin Res Cardiol
ejection fraction (LVEF) [7], cardiac biomarkers [8] such as brain natriuretic peptides (BNP) [9], troponins [10], soluble ST2 [11], Galectin-3 [12], high-mobility-group box 1 [13], heart rate at rest [14] or immunohistological signs of inflammation [15]. However, due to the diverse presentation of myocarditis and the widely varying individual course of progression of the disease, early and accurate diagnosis of myocarditis and identification of novel reliable risk factors that help to confirm the diagnosis of myocarditis and identify high-risk patients are still warranted. Chemokines interacting with their receptors play a crucial role in the recruitment of inflammatory cells to myocarditis lesions and are of central importance for initiation, maintenance and resolution of inflammation [16]. Recent studies demonstrated cardiodepressive effects of a number of chemokines including CX3CL1/fractalkine [16]. SDF-1, also known as CXCL12, is an inflammatory CXC-chemokine that is rapidly and transiently upregulated in response to tissue injury [17]. In the heart, SDF-1 is expressed by stromal cells, endothelial cells, fibroblasts, dendritic cells and cardiomyocytes [18, 19]. SDF-1 binds to its receptors CXCR4 and CXCR7 regulating adhesion and migration of inflammatory cells including platelets, monocytes and lymphocytes [20– 22]. SDF-1 further increases homing of circulating CXCR4positive progenitor cells to ischemic tissue [23], and plays a major role in cell proliferation and ventricular remodeling in ischemic cardiomyopathy [19, 24]. We and other groups were able to show that SDF-1 is cardioprotective after myocardial hypoxemia and increases recruitment of circulating progenitor cells promoting angiogenesis and supporting myocyte survival [22, 24, 25]. In recent studies, we observed an association between platelet SDF-1 expression and reduced left ventricular function due to ischemic cardiomyopathy [26], as well as the number of circulating CD34? progenitor cells and platelet-progenitor cells co-aggregates augmenting recruitment of CD34? cells into ischemic microcirculation [25, 27]. Additionally, it was recently shown that increased plasma SDF-1 levels were associated with development of HF and all-cause mortality [28]. However, the role of the inflammatory chemokine SDF-1, which is transiently expressed in the myocardium upon cardiomyocyte damage, in myocarditis is still unknown. We therefore explored whether endomyocardial expression of SDF-1 may serve as predictor of mortality in a cohort of patients undergoing endomyocardial biopsy for suspected myocarditis.
Materials and methods Patients We prospectively included 174 consecutive patients who underwent endomyocardial biopsy (EMB) between 3/2009
123
and 6/2011 as part of an evaluation for suspected myocarditis at our university center. Indications for EMB were based on the current guidelines [29]. Patients were included if they clinically presented with signs of possible myocarditis such as history of recent infection, impaired global or regional left ventricular function, increase in serum concentrations of cardiac biomarkers, pericardial effusion, sustained or non-sustained ventricular tachycardia of unknown origin. Patients with significant coronary artery disease as the underlying cause of HF were excluded. All patients received medication according to current ESC and ACC/AHA guidelines depending on their LVEF and HF symptoms [30]. The study was approved by the local ethics committee. Informed consent was obtained from all patients. Assessment of clinical risk factors NYHA functional class was assessed by experienced cardiologists at study entry. LVEF was estimated by echocardiography (iE33, Philips Medical Systems) by modified Simpson rule with images obtained from apical 4and 2-chamber views. Laboratory studies included BNP and troponin I (TnI). BNP and TnI levels were assessed by immunoassay (ADVIA Centaur BNP Assay and ADVIA Centaur TnI-Ultra, Siemens Healthcare Diagnostics, respectively). Patients were classified as BNP-positive or TnI-positive if BNP level was above 100 ng/L or TnI level above 0.04 lg/L, respectively. Analysis of endomyocardial biopsies Approximately five EMB specimens were taken with a dedicated bioptome (Biopsy Forceps, Cordis Corporation, USA) from the right ventricular septum. Three biopsy specimens were fixed in 4 % buffered formaldehyde for immunohistology. Two tissue samples were fixed in RNAlater (Ambion Inc., Foster City, USA) for detection of viral genomes by nested real time polymerase chain reaction (RTPCR). Paraffin-embedded EMB were stained with Masson’s trichrome reflecting myocyte necrosis, as well as interstitial fibrosis and analyzed by light microscopy [31, 32]. Histological analysis followed the Dallas criteria [32] complemented by immunohistology to assess inflammation. According to the Dallas criteria [32], inflammatory cardiomyopathy was defined by lymphocytic infiltrates in association with myocyte necrosis. According to the WHO/ International Society and Federation of Cardiology Task Force on the Definition and Classification of Cardiomyopathies [33], inflammation was considered present after immunohistological detection of mononuclear infiltrates with [14 leucocytes per 1 mm2 in the myocardium, in
Clin Res Cardiol
addition to enhanced expression of HLA class II molecules. EMB without signs of inflammation but with myocardial damage and fibrosis were classified as non-inflammatory cardiomyopathy. Those patients suffered predominantly from idiopathic dilative cardiomyopathy or hypertensive heart disease. According to established scores for evaluation of CD68 and MHCII [1, 15, 34, 35], sections stained for SDF-1 were analyzed by a semiquantitative score ranging from 1 (no expression) to 4 (ubiquitous high expression) by two coinvestigators in a blinded manner from one to two sections for each staining [36]. Fleiss statistics revealed a free marginal kappa of 0.7 for SDF-1 indicating a good agreement between the operators. EMB stained for SDF-1 were then classified in a dichotomized way as ‘‘negative’’ (no/ low) or ‘‘positive’’ (moderate/high expression). The extent of myocardial fibrosis in EMB was defined as an index as described previously [37]. The percentage area of fibrosis in the section was evaluated by dividing the sum of the fibrotic areas of the section by that of the total tissue area. According to this fibrosis index, patients were classified as having no/mild, moderate, or severe fibrosis. Follow-up and study endpoints 132 of the 174 patients presented in our outpatient clinics for follow-up visits scheduled at 6-months intervals. For the remaining 42 patients who were not seen in our clinics, telephone contact was made to determine occurrence of an endpoint. None of the patients were lost to follow-up. The primary study endpoint was death from all causes. The secondary endpoint was defined as the composite of the rate of death from all causes and HF-related rehospitalizations. Physicians who were in charge of decision for rehospitalizations were not involved in the study. Statistical analysis Continuous variables are expressed as median and interquartile range (IQR) and compared using Mann–Whitney U test. Categorical data are presented as proportions and analyzed by Chi square test. The following risk factors were prospectively assessed: NYHA functional class, LVEF, BNP, TnI and SDF-1. Continuous parameters were dichotomized at established cut-off values. Multivariable analyses were performed using a Cox proportional hazards model with the enter procedure, and adjusted to age and gender. Predefined subgroup analyses were performed for patients with inflammatory and non-inflammatory cardiomyopathy. Survival curves of patients grouped by prespecified variables were calculated by Kaplan–Meier analyses and compared with log-rank test. Time point for beginning of survival analysis was the date of EMB. As a
measure of agreement between the different blinded raters of EMB staining, Fleiss’ kappa coefficient was calculated [38]. A receiver-operator characteristic (ROC) curve for prediction of mortality was constructed for SDF-1 by plotting the dependency of specificity on sensitivity. The ROC curve was quantified by the integral of the curve [area under the curve (AUC)]. Confidence intervals (CIs) were estimated using bootstrapping. Correlations between SDF1 and fibrosis, as well as with CD3, MHCII and LVEF were tested with Spearman rank correlation. Comparisons were considered statistically significant if two-sided p value was \0.05. Statistical analyses were performed using SPSS software version 21 (SPSS Inc.).
Results Baseline characteristics of the study cohort are presented in Table 1. Patients were rather young [median age 53 (IQR 45–67) years] and predominantly males (76 %). 83 % of the patients had HF symptoms (NYHA functional class CII). LVEF was impaired with \40 % in 51 % of the patients. 75 and 29 % of patients presented with elevated serum concentrations of BNP and TnI, respectively. 73 % of the EMBs (n = 127) showed myocardial inflammation. 8/127 patients presented with histopathological and immunohistological signs of acute myocarditis and the remaining 119/127 patients with chronic myocarditis. 64 of the 174 patients (36.8 %) were virus-positive. From those patients, 34/174 (19.5 %) were parvovirus B19 (PVB19) positive, 19/174 patients (10.9 %) were Epstein Barr virus (EBV) positive, 17/174 patients (9.8 %) were human herpesvirus 6 (HHV6) positive and 1/174 (0.6 %) coxsackie B3 (CVB3) positive. Three patients had a co-infection of PVB19 and HHV6, three patients PVB19 and EBV, and one patient EBV as well as HHV6. 53 % of the patients had a SDF-1 positive EMB. SDF-1positive patients had a higher prevalence of myocardial inflammation (90 vs. 54 %, p \ 0.0001) with higher amounts of CD3? T cells (58 vs. 16 %, p \ 0.0001), CD68? macrophages (76 vs. 18 %, p \ 0.0001) and enhanced expression of MHCII (76 vs. 18 %, p \ 0.0001), when compared to SDF-1 negative patients (Fig. 1b). Furthermore, patients with SDF-1 positive EMB were significantly more often virus-positive (45 vs. 28 %, p = 0.024) and TnI-positive (37 vs. 20 %, p = 0.011). In EMB of patients with myocardial inflammation, SDF-1 was predominantly present in endothelial cells and interstitial cells, most likely representing fibroblasts and immune cells, e.g. macrophages and dendritic cells (Fig. 1a). SDF-1 was not expressed in the healthy human myocardium of control subjects (Fig. 1a). Of note, all patients with acute myocarditis (100 %) and 75/119 (63.0 %) of
123
Clin Res Cardiol Table 1 Baseline characteristics of the cohort of patients with suspected myocarditis All patients (n = 174)
Survivors (n = 154)
Non-survivors (n = 20)
p value
Demographics Age (years)
53 (45–67)
Males (%)
133 (76.4) 2
Body mass index (kg/m )
25 (23–29)
53 (44–66) 118 (76.6) 26 (24–29)
62 (49–71)
0.123
15 (75.0)
0.872
24 (22–29)
0.112
Risk variables NYHA class CII (%)
144 (82.8)
128 (83.1)
16 (80.0)
0.728
88 (50.6)
73 (47.4)
15 (75.0)
0.020
LVEF \40 % BNP-positivea (%)
131 (75.3)
112 (72.7)
19 (95.0)
0.030
Troponin I positivea (%)
50 (28.7)
40 (26.0)
10 (50.0)
0.025
PAPsys (mmHg)
24 (20–35)
24 (20–33)
32 (21–41)
0.047
Results of endomyocardial biopsy (%) Inflammatory CMP 127 (73.0)
112 (72.7)
15 (75.0)
0.829
Non-inflammatory CMP
47 (27.0)
42 (27.3)
5 (25.0)
0.829
Virus-positive
64 (36.8)
57 (37.0)
7 (35.0)
0.861
SDF-1-positive
92 (52.9)
74 (48.1)
18 (90.0)
