P6353Iron depletion in human cardiomyocytes cultured with sera from myocarditis patients
Authors: Kobak K A , Schubert J , Kasztura M , Franczuk P , Dziegala M , Drozd M , Tkaczyszyn M , Kulej-Lyko K , Bania J , Banasiak W , Ponikowski P , Jankowska E A 
Published: October 2019

Abstract

Background
Cardiac myocytes, which are particularly sensitive to disordered iron homeostasis, are the main cells affected in the course of myocarditis. Iron is essential for the proper maintenance of energy metabolism but also plays key role in inflammation and ROS production. We hypothesize that iron homeostasis might be involved in the pathophysiology of myocarditis.

Purpose
The aim of the study was to assess differences in the expression of key genes and proteins involved in iron homeostasis, cardiac malfunctioning, and protection against ROS in human cardiomyocytes (HCMs) cultured in the indirect model of myocarditis.

Methods
HCMs were cultured for 48 hours with 10% of sera from patients with acute myocarditis (n=11) and after 6 weeks of recovery, and also with sera from healthy controls (n=7). We analyzed expression of light and heavy ferritin chains [FTL, FTH], transferrin receptor 1 [TfR1], galectin 3 [LGALs3], TGFβ signaling [TGFβ1, TGFβ2, TGFβ3], glutathione peroxidase [GPX] and superoxide dismutase [SOD1] at the mRNA level using RTqPCR and at the protein level using Western bloting. We compared obtained data with the clinical characteristics of patients.

Results
In HCMs exposed to sera from myocarditis patients, in comparison to those treated with sera from healthy controls, we found a significant increase in an expression of TfR1 both at mRNA and protein level (p<0,01). We also observed that elevated expression of TfR1 in cells correlated with serum levels of total iron (R=-0,52; p<0,05), CRP (R=0,67; p<0,05), and NT-proBNP (R=0,55; p<0,05), suggesting increased iron demand in HCMs and its possible relation to inflammation and hemodynamic dysfunction in patients.
Moreover, we observed elevated expression of FTH and FTL at the mRNA level (both p<0,01) and its strong correlation with expression of TfR1 as well as with increased levels of CRP in sera. It could be explained by the double role of ferritin in iron storage and in inflammation.
Interestingly, we noticed detrimental effects of myocarditis sera on HCMs reflected by augmented expression of galectin 3 (p<0,01) and disturbances in TGFβ genes, in comparison to those treated with sera from healthy controls. Augmented expression of galectin 3 was strongly related to disturbed iron homeostasis, manifesting itself by correlations with TfR1 (R=0,77; p<0,05), FTH (R=0,92; p<0,05) and FTL (R=0,76; p<0,05).
In addition, HCMs treated with sera from myocarditis patients showed an increase in expression of ROS protective genes such as SOD1 and GPX (both p<0,01), indicating higher oxidative stress in these cells.
We noticed that gene expression profile was similar in HCMs treated with sera collected after 6 weeks of clinical recovery, suggesting that the negative impact of sera was preserved.

Conclusions
Malfunctioning of cardiomyocytes in course of myocarditis might be related to disturbances in the iron homeostasis.

Acknowledgement/Funding
The present study was financially supported by the National Science Centre (Krakow, Poland; grant no. 2014/13/B/NZ5/03146)