Inflammation, iron and vitamin D metabolism in different cardiomyopathy aetiologies

Immune activation coincides with disturbances in iron and vitamin D metabolism in patients with cardiomyopathy. In this study, we investigated whether there are differences regarding immune activation, iron and vitamin D metabolism between the different cardiomyopathy aetiologies. Patients and methods: Parameters of iron metabolism (haemoglobin, iron, transferrin, transferrin saturation, ferritin, hepcidin), vitamin D metabolism (Ct-FGF23, parathormone, phosphate, vitamin D) and immune activation (C-reactive protein and neopterin) were determined in 149 patients (98 men, 51 women) with non-ischaemic cardiomyopathy. Results: Patients with amyloid cardiomyopathy presented with higher neopterin, ferritin and hepcidin levels than other cardiomyopathy aetiologies. Furthermore, they showed the highest rate of cardiovascular events. C-reactive protein levels were significantly higher in patients with inflammatory cardiomyopathy. Patients with virus positive cardiomyopathy presented with significantly higher ferritin and Ct-FGF23 levels compared to patients with virus negative inflammatory cardiomyopathy. Conclusion: This study indicates that there are some differences regarding the extent of immune activation and inflammation as well as alterations in iron metabolism disorders between different cardiomyopathy aetiologies. Further studies with larger patient cohorts are needed to investigate these findings more precisely.


Introduction
Cardiomyopathies (CMPs) represent a heterogenic group of heart muscle disease. The heart muscle can be affected as a primary pathologic process or secondarily in the course of a systemic disorder. CMPs can be classified due to their morphology into dilated CMP (DCM), hypertrophic CMP (HCM), restrictive CMP (RCM), arrhythmogenic right ventricular CMP (ARVC) and undefined CMP [1][2][3]. DCM is the most common morphologic form of CMP with rising incidence and defined as dilatation of the left ventricle (LV) and later on of both ventricles [4]. HCM is the most common genetic heart disease and the most common cause of sudden cardiac death in young adults [5]. Its morphology is characterised by a cardiac hypertrophy especially of the LV [6]. RCM is the most uncommon morphologic form of CMPs in developed countries and characterised by an increased myocardial stiffness reflected by diastolic dysfunction usually of the LV [7]. ARVC is a relatively uncommon genetic heart disease that typically affects men between the second and fourth decade of life and is associated with loss of right ventricular myocardium with following substitution by fibrous and fatty tissue [8].
We know that immune activation plays an important role in different CMP aetiologies (e.g. inflammatory, metabolic or toxic CMP) [26]. Immune activation is also related to alterations of iron metabolism [27]. Actually, both iron deficiency and iron overload affect the CMP pathophysiology [28]. Iron deficiency reduces exercise tolerance [29] and leads to progressive heart failure and increased mortality [30], while iron overload and consequently increased oxidative stress induces CMP [31]. Finally, the recently detected FGF23 also effects the heart function [32] and interacts with the immune system and iron metabolism [33]. Since immune activation as well as alterations in iron and vitamin D metabolism effect heart function, we were interested, whether these mechanisms differ between patients with different CMP morphologies and aetiologies.

Study population
Within this retrospective study, we analysed a data set that initially included 475 Caucasian patients with heart failure (HF) due to nonischaemic cardiomyopathy (CMP), who underwent right heart catheterization between 2009 and 2014 in the Cardiology department of the Innsbruck University Hospital. We only included data of 149 patients (98 men, 53 women) for further statistical analyses, since data regarding biomarkers of immune activation and inflammation (CRP, neopterin), iron metabolism parameters (haemoglobin, iron, ferritin, transferrin, transferrin saturation) and vitamin D metabolism parameters (including C-terminal fibroblast growth factor 23 (Ct-FGF23) concentrations) were not available for all patients. Acute HF, coronary artery disease on coronary angiography or vitamin D or calcium supplement within the last 6 months were also exclusion criteria. The diagnosis of HF was made due to the presence of current or previous symptoms or characteristic clinical signs and evidence of LV dysfunction. Patients were treated according to CHF guidelines.
Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance with tenets of the Helsinki Declaration, and has been approved by the local ethics committee of Innsbruck Medical University (ID of the Ethical votum: UN4280, session number 298/4.11).
Informed consent: Informed consent has been obtained from all individuals included in this study

Follow-up analysis
The event-free survival was defined as period of time between the first hospitalisation and the combined endpoint, which was either a heart transplantation (HTx), a ventricular assist device (VAD) implantation, a re-hospitalisation for cardiac decompensation or patients' death. Cut-off date for the follow-up was May 2017. Information about those events were received from the clinical information system (KIS), the local mortality registry, from the patients' relatives or from the patients themselves.

Measurements
Fasting blood samples were drawn in all patients at study entry and stored at -80°C. All routine laboratory variables were measured at our central laboratory that undergoes regular internal and external quality audits. Neopterin was measured by an enzyme-linked immunosorbent assay (ELISA; IBL International GmbH, Hamburg, Germany) and the C-reactive protein (CRP) by an immunoturbidimetry test (Roche, Mannheim, Germany). Creatinine and NT-proBNP were determined by standardized automated tests. Hepcidin-25 levels were determined by ELISA (DRG Instruments GmbH, Marburg, Germany) in 91 patients. Iron levels were measured with the FerroZine TM method without deproteinization (Merck KGaA, Darmstadt, Germany). Ferritin levels were detected with an immunoturbidimetry test containing anti-ferritin antibodies from rabbits (Roche, Mannheim, Germany). Transferrin levels were also detected with an immunoturbidimetry test containing specific antibodies from rabbits (Roche, Mannheim, Germany). The transferrin saturation (TSAT) was calculated as followed: iron / transferrin x 70.9. Haemoglobin levels were analysed photometrically at 555 nm (XE-5000, Sysmex GmbH, Wien, Austria). FGF23 was determined using an FGF23 assay (Immutopics Inc., San Clemente, CA, USA; interassay coefficient of variation <5%) that detects epitopes within the carboxyl-terminal domain of FGF23 (Ct-FGF23) with polyclonal antibodies. Therefore, the test used in this study detects both cFGF23 and iFGF23. Circulating concentrations of Ct-FGF23 are expressed as relative units per millilitre (RU/mL). Glomerular filtration rate (GFR) was estimated using the IDMS-traceable MDRD Study equation ( The cardiac output (CO) was measured in course of a left heart catheterisation and the left ventricular ejection fraction (LV-EF) was measured by echocardiography.

Statistical analysis
Data are presented as median (25 th , 75 th percentile) or n (%), as appropriate. Shapiro-Wilks test was used to test for normality in distribution of included parameters. Nonnormally distributed parameters are expressed as median with interquartile range (IQR) and were compared by non-parametric tests (Mann-Whitney U, Kruskal-Wallis). Outcome differences between classified subgroups were analysed with the Log-rank test.
P-values < 0.05 were considered to indicate statistical significance. Statistical analysis was performed using SPSS 24.0 for Macintosh (IBM Corp., Armonk, NY, USA).  Table 1.

Patient characteristics: NYHA classification
Patients with RCM had a preserved LV-EF over 50%, were older and tended to have the highest NT-proBNP levels. Patients with DCM had the lowest LV-EF, and a higher cardiac output (CO) compared to patients with RCM. The highest neopterin concentrations were found in patients with RCM, while those patients had the lowest CRP concentrations. ( We also divided the inflammatory aetiology into virus negative (n = 69, 81.2 %), virus positive (n = 10, 11.8 %) and postinflammatory (n = 6, 7. Demographic and epidemiologic data, laboratory measurements and haemodynamics within the aetiological subgroups are listed in Table 2.
Patients with amyloid CMP had significantly higher neopterin levels (9.

Iron metabolism and cardiomyopathy aetiologies
Patients with RCM had the lowest TSAT and iron levels as well as the highest transferrin levels. Haemoglobin levels were the highest in patients with DCM and the lowest in patients with undefined CMP ( Table 1).
We also investigated parameters of iron metabolism in patients with inflammatory CMP subgroups: results are depicted in Figure 2 -significant differences between the groups are marked.
Moreover, we analysed for differences of iron metabolism parameters between patients within and without amyloid CMP: results are depicted in Figure 3 significant differences are marked.
No differences concerning iron metabolism parameters were detected between patients with (active or chronic or both) or without signs of myocarditis in the EMB. However, patients with signs of myocarditis tended to have lower ferritin concentrations compared to patients without signs of myocarditis in the EMB (131 µg/L [57 -259] vs. 191 µg/L [89 -291], p = 0.066).

Vitamin D metabolism and cardiomyopathy aetiologies
The highest Ct-FGF23 concentrations were found in patients with RCM ( Table 1). Patients with inflammatory CMP had lower Ct-FGF23 concentrations (20.9 RU/mL [13.6 -37.7] vs. 32.5 RU/mL [14.8 -58.3], p = 0.136). Other vitamin D metabolism parameters showed no differences between the CMP aetiologies. In addition, there were no differences of vitamin D metabolism parameters between patients with or without sings of myocarditis in the EMB (active, chronic and both).

Outcome analysis and cardiomyopathy aetiologies
Patients with RCM had a three-fold higher event-rate (63.1 %) compared to patients with DCM (22.3 %). The event-rate of patients with HCM was also nearly twice as high (38.7 %) compared to patients with DCM (p = 0.001, Figure 4A).
Patients with amyloidosis showed the highest event rate after 5 years (72.2 % vs. 20.6 % in patients with inflammatory CMP and 30.1 % in patients with idiopathic CMP all p < 0.001, Figure 4B).

Discussion
Amyloid CMP represents as RCM with a rapidly progressive and fatal pathogenesis. This is confirmed by our study, since patients with amyloid CMP, which appears as RCM, as well as patients with RCM had the highest event rate after 5 years. Recent studies demonstrated, that amyloidosis triggers inflammatory reactions [35] and is associated with increased systemic and cardiac oxidative stress [36][37][38]. T-helper cell type 1 (TH 1 ) immune activation results in the activation of macrophages, which produce neopterin concomitantly to reactive oxygen species (ROS) [39][40][41]. In our study, neopterin levels were found to be significantly higher in patients with amyloid CMP compared to patients with other CMP aetiologies. Moreover, significantly higher ferritin and hepcidin levels, which are both conditions that are seen in patients with a TH 1 immune activation [27], were found in patients with amyloid CMP compared to patients with other CMP aetiologies. This indicates that TH 1 immune activation with recruitment of activated macrophages into the myocardium as a consequence of deposited amyloid proteins might be a more important pathophysiological mechanism compared to other CMP aetiologies and goes along with changes of the iron storage capacity. Nevertheless, patients with amyloid CMP do not show decreased serum iron levels or a low TSAT as one would expect in patients with fully developed anaemia of chronic disease (ACD) [27]. Interestingly, patients with amyloid CMP also had the lowest CRP concentrations -which is in line with the notion that elevated CRP is associated with TH 2 rather than TH 1 immune response. Patients with amyloid CMP were also the oldest patients with a median age of approximately 70 years, which is in accordance with actual literature [25]. Further studies with larger patient cohorts will be needed to investigate the impact of inflammation and changes of iron metabolism on the pathophysiology of amyloid CMP.
Contrary to patients with amyloid CMP, patients with inflammatory CMP had higher CRP levels compared to patients with no inflammatory CMP, while neopterin levels did not differ. Interestingly, there were no differences of median CRP and neopterin levels between patients with virus positive and virus negative inflammatory CMP as well as between patients with and without signs of myocarditis in the EMB (both acute and/or chronic). This is in contrast to previous studies, which described higher neopterin levels in patients with inflammatory CMP, especially in patients with active myocarditis [42][43][44]. Since immune activation goes along with increased ferritin levels (ferritin is inter alia an acute phase protein), significantly higher ferritin levels in patients with inflammatory CMP might be expected [45]. Still, in our study patients with other CMP aetiologies had higher ferritin levels than patients with inflammatory CMP. Inflammation is known to be a leading cause of iron metabolism disturbances; however, other CMP aetiologies may trigger additional pathophysiological mechanisms. Nevertheless, when comparing patients with virus negative and virus positive inflammatory CMP, the latter showed significantly higher ferritin levels. Interestingly, there were no differences concerning iron metabolism parameters between patients with (active or chronic or both) or without signs of myocarditis in the EMB.
Ct-FGF23 levels were also lower in patients with virus positive inflammatory CMP compared to patients with virus negative inflammatory CMP and with no inflammatory CMP. Further longitudinal studies will be needed to better characterize the role of inflammation and immune-mediated disturbances of iron and vitamin D metabolism in patients with different kinds of CMP.

Strengths and limitations
Because of the low number of patients within some CMP aetiology subgroups and the fact that neopterin, Ct-FGF23 and iron metabolism parameters were not available of all patients initially included in the study, the findings of a small patient cohort do not allow unrestricted generalisation for all cardiomyopathy patients. The small number of patients within some CMP aetiology subgroups did not allow us to analyse for gender-differences in an appropriate way. As we also did not have data of all patients' co-morbidities and potential risk factors such as smoking or alcohol consumption studies including those factors would also be of interest.

Conclusion
This study indicates that the extent of inflammation varies in patients with CMP of different aetiologies. Furthermore, we could find changes of iron and vitamin D metabolism in CMP, which are worth being investigated in more detail in larger patient cohorts.
Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or notfor-profit sectors.