Stephan Baldus / Martin Mollenhauer / Volker Rudolph - B 2

Inflammatory pathways propagating ventricular tachycardia - the role of myeloperoxidase for postischemic pro-arrhythmic remodeling

Ventricular tachycardia after ischemic heart disease is a major cause of mortality in western countries with sparse treatment options. This study investigates the importance of the leukocyte derived enzyme myeloperoxidase (MPO) for ventricular pro-arrhythmic remodeling following myocardial infarction in mice.We now could show that MPO contributes to the development of induced and spontaneous ventricular tachycardias (VT) by disrupting the homogenous ventricular conduction in two murine models of ischemic cardiomyopathy.

Introduction

Among the several mechanisms linked to ventricular arrhythmia induction following ischemic myocardial damage, electrical reentry has evolved as particularly important. It involves areas of functional conduction block, around which electrical conduction travels to leave the initially excited area enough time to regain excitability. A variety of structural and functional alterations have been suggested as underlying mechanisms: fibrosis, reduction and functional impairment of connexins and gap-junctions and decreased cellular ion currents. In this regard MPO gains potential significance since: a) MPO is secreted and lastingly activated in the area at risk following myocardial ischemia and b) based on its prooxidant properties – MPO is potentially involved in structural and functional alterations yielding ventricular arrhythmia.

Here in, we subjected wild-type (WT) and MPO-deficient (Mpo-/-) mice to two models of ischemic cardiomyopathy, myocardial ischemia/reperfusion damage (I/R) and permanent coronary artery occlusion (PI) and investigated MPO-dependent effects on connexin 43 (Cx43) stability, which is a major myocardial gap junction protein responsible for electrical coupling, in vivo and in vitro.

Total Cx43 in the infarct- and periinfarct-zone

Immunostainings for Cx43 revealed a complete absence of Cx43 immunoreactivity in the infarct zone of WT and Mpo-/- animals (not shown). In the periinfarct region, a significantly decreased signal for Cx43 was detected in WT I/R mice compared to sham-operated mice. In contrast, no reduction was recognized in Mpo-/- I/R mice in comparison to sham-operated Mpo-/- mice (Figure 1A upper panel, 1B). Strikingly, Mpo-/- hearts revealed significantly higher immunoreactivity for Cx43 in the periinfarct region 3 days and 21 days after PI induction as compared to WT hearts (Figure 1A lower panel, 1C). In both models, I/R and PI, ventricular Cx43 mRNA expression levels did not differ between WT and Mpo-/- animals pointing towards a posttranslational effect of MPO on Cx43 levels (Figure 1D).

MMP-7 depended Cx43 degradation

Studies in isolated adult cardiomyocytes confirmed the decreased content of Cx43 following incubation with MPO. Strikingly, this effect was partly reverted by additional treatment with an inhibitor of MMP-7, an enzyme not only shown to directly bind and degrade Cx43 following myocardial ischemia but also to be activated by MPO-derived hypochlorous acid (Figure 2A). In addition, activation of MMP-7 was significantly more abundant in the periinfarct region of WT compared to Mpo-/- mice following I/R in vivo (Figure 2B).

To further investigate the effect of MPO mediated Cx43 degradation via MMP-7 conduction patterns of a monolayer of induced pluripotent stem cell-derived cardiomyocytes (iPSCM), which are devoid of MMP-7, were assessed by in vitro mapping analyses. Spontaneously beating iPSCM showed homogeneous conduction patterns (Figure 2C) under control conditions. Addition of MPO/H2O2 or pro-MMP-7 alone had no effect on the observed patterns. However, concomitant treatment with pro-MMP-7/MPO/H2O2 resulted in a severe disruption of conduction homogeneity as demonstrated by an increased inhomogeneity index (Figure 2D) an elevated absolute inhomogeneity (Figure 2E) and an increased variation coefficient (Figure 2F), indicative of diminished intercellular electrical coupling among iPSCM. Conversion of pro-MMP-7 to active MMP-7 by MPO-derived HOCl has been described before. Accordingly, incubation of the cardiac muscle cell line HL-1, which also lacks MMP-7 protein expression, with MPO/H2O2/pro-MMP-7 resulted in decreased Cx43 levels, whereas single treatment with MPO/H2O or pro-MMP-7 did not induce Cx43 degradation (not shown).

Perspectives

The current data point towards an important causal role of MPO in myocardial electrical remodeling in two different murine models of ischemic injury. Mechanistically, MPO not only disturbs electrical conduction by structural fibrotic remodeling but also by impairment of Cx43 integrity through the activation of MMP-7. Ongoing experiments will further disclose whether MPO modulates Ca-homeostasis and ion channel function in mice subjected to ischemic injury. These results not only indicate that the innate immune system and namely leukocytes exert proarrhythmogenic properties, but also point toward myeloperoxidase as a potential pharmacological target in this disease.
Therefore, we will apply pharmacological MPO inhibition in vivo and in vitro.

Selected publications

1. Rudolph, V., Andrié, R. P., Rudolph, T. K., Friedrichs, K., et al. (2010). Myeloperoxidase acts as a profibrotic mediator of atrial fibrillation. Nat Med 16, 470-474 

 2. Mollenhauer, M. et al. (2017) Myeloperoxidase Mediates Postischemic Arrhythmogenic Ventricular Remodeling. Circ Res. 2017 Jun 23;121(1):56-70.


Prof. Dr. Stephan Baldus

Dept. III of Internal Medicine / RG location - LFI Building

Prof. Dr. Stephan Baldus

Principal Investigator B 2
Executive Board Member

stephan.baldus@uk-koeln.de

Work +49 221 478 32512

Fax (Work) +49 221 478 32512

Heart Center Dept. III of Internal Medicine
Kerpener Str. 62
50937 Cologne

http://herzzentrum.uk-koeln.de/de/kardiologie

Publications - Stephan Baldus

Link to PubMed


Dr. Martin Mollenhauer

Dept. III of Internal Medicine / RG location - LFI Building

Dr. Martin Mollenhauer

Co-Principal Investigator B 2

martin.mollenhauer@uk-koeln.de

Work +49 221 478 87445

Heart Center University Hospital Cologne
Kerpener Str. 62
50937 Cologne

Publications - Martin Mollenhauer

Link to PubMed


Prof. Dr. Volker Rudolph

Dept. III of Internal Medicine / RG location - LFI Building

Prof. Dr. Volker Rudolph

Co-Principal Investigator B 2

volker.rudolph@uk-koeln.de

Work +49 221 478 32495

Fax (Work) +49 221 478 32400

Heart Center University Hospital Cologne
Kerpener Str. 62
50937 Cologne

http://herzzentrum.uk-koeln.de/de/kardiologie/forschung/ag_rosenkranz

Publications - Volker Rudolph

Link to PubMed

Group Members

Sakine Simsekyilmaz (PhD student)
Matti Adam (Medical doctor)
Dennis Mehrkens (Medical doctor)
Simon Braumann (Medical doctor)
Alexander Hof (Medical doctor)
Felix Nettersheim (Medical doctor)
Senai Bokredenghel (Medical doctor)
Max Wißmüller (Medical doctor)
Karen Gerke (Medical student)
David Muders (Medical student)
Malte Kochen (Medical student)
Julian Lemties (Medical student)
David Schlüter (Medical student)
Simon Geißen (Medical student)
Birgit Arand (Medical student)
Johannes Dohr (Medical student)
Gülsah Duman (Medical student)
Nam Guy Im (Medical student)
Lisa Remane (Medical student)
Simon Grimm (Technician)
Nadja Kulesza (Technician)
Christina Schroth (Technician)
Iris Berg (Study nurse)

Figure 1

CMMC Research Odenthal

Figure 1. Connexin43 (Cx43) integrity.
(A) Immunoreactivity for Cx43 and N-cadherin in the cardiac periinfarct zone of WT and Mpo-/- subjected to sham-operation, 7 days I/R or 3- and 21 days PI (red=N-cadherin, green=Cx43, blue=DAPI; scale bar=30 µm). (B) Quantitative analysis ofCx43 immunoreactivity in the periinfarct zone of WT- and Mpo-/- mice upon I/R or (C) 3- and 21 days PI. (D) Analysis of left ventricular Cx43 mRNA expression of sham, I/R or PI hearts of WT and Mpo-/- mice.

Figure 2

CMMC Research Odenthal

Figure 2: MPO-dependent effect of MMP-7 on Cx43 and electrical homogeneity in iPSCM.
(A) Immunoblot analyses of Cx43 in isolated cardiomyocytes upon MPO- with and without additional MMP-inhibitor treatment. (B) MMP-7 activity in the periinfarct region of I/R WT and Mpo-/- hearts.
(C) Representative maps of spontaneous conduction of iPSCM treated with NaCl (control), MPO/H2O2, pro-MMP-7 or MPO/H2O2/pro-MMP-7 with analysis of
(D) inhomogeneity index,
(E) enhanced absolute inhomogeneity and (F) variation of velocity.