Myeloperoxidase Mediates Postischemic Arrhythmogenic Ventricular RemodelingNovelty and Significance
Rationale: Ventricular arrhythmias remain the leading cause of death in patients suffering myocardial ischemia. Myeloperoxidase, a heme enzyme released by polymorphonuclear neutrophils, accumulates within ischemic myocardium and has been linked to adverse left ventricular remodeling.
Objective: To reveal the role of myeloperoxidase for the development of ventricular arrhythmias.
Methods and Results: In different murine models of myocardial ischemia, myeloperoxidase deficiency profoundly decreased vulnerability for ventricular tachycardia on programmed right ventricular and burst stimulation and spontaneously as assessed by ECG telemetry after isoproterenol injection. Experiments using CD11b/CD18 integrin–deficient (CD11b−/−) mice and intravenous myeloperoxidase infusion revealed that neutrophil infiltration is a prerequisite for myocardial myeloperoxidase accumulation. Ventricles from myeloperoxidase-deficient (Mpo−/−) mice showed less pronounced slowing and decreased heterogeneity of electric conduction in the peri-infarct zone than wild-type mice. Expression of the redox-sensitive gap junctional protein Cx43 (Connexin 43) was reduced in the peri-infarct area of wild-type compared with Mpo−/− mice. In isolated wild-type cardiomyocytes, Cx43 protein content decreased on myeloperoxidase/H2O2 incubation. Mapping of induced pluripotent stem cell–derived cardiomyocyte networks and in vivo investigations linked Cx43 breakdown to myeloperoxidase-dependent activation of matrix metalloproteinase 7. Moreover, Mpo−/− mice showed decreased ventricular postischemic fibrosis reflecting reduced accumulation of myofibroblasts. Ex vivo, myeloperoxidase was demonstrated to induce fibroblast-to-myofibroblast transdifferentiation by activation of p38 mitogen-activated protein kinases resulting in upregulated collagen generation. In support of our experimental findings, baseline myeloperoxidase plasma levels were independently associated with a history of ventricular arrhythmias, sudden cardiac death, or implantable cardioverter–defibrillator implantation in a cohort of 2622 stable patients with an ejection fraction >35% undergoing elective diagnostic cardiac evaluation.
Conclusions: Myeloperoxidase emerges as a crucial mediator of postischemic myocardial remodeling and may evolve as a novel pharmacological target for secondary disease prevention after myocardial ischemia.
Sudden cardiac death is the leading cause of death after myocardial infarction, and three quarters of patients dying from an acute arrhythmic event are diagnosed for coronary artery disease.1 Myocardial ischemia is one of the most powerful triggers of leukocyte recruitment and activation. In particular, neutrophils are among the first cells found in the area at risk, and their activation in the setting of acute myocardial ischemia can be measured systemically. On activation, neutrophils release myeloperoxidase, a heme enzyme abundantly expressed in these cells. Myeloperoxidase until recently was solely viewed as part of the innate immune defense.2 However, myeloperoxidase has been shown to promote potent proinflammatory vascular properties, facilitating the consumption of endothelial nitric oxide directly or by generation of potent reactive oxygen species.3 Myeloperoxidase has been demonstrated to be involved in myocardial remodeling after myocardial injury. It increased myocardial collagen deposition after ligation of the left anterior descending (LAD) artery,4 and myeloperoxidase-deficient (Mpo−/−) mice exhibited less left ventricular (LV) dilatation and attenuated impairment in systolic LV function.5 In the atria, myeloperoxidase also increased fibrotic remodeling, which was linked to an increased susceptibility to atrial fibrillation.6 Apart from ventricular fibrosis, there is indirect evidence that myeloperoxidase promotes the degradation of the gap junctional protein Cx43 (Connexin 43), which has been firmly linked to ventricular arrhythmogenesis. Thus, Cx43 availability has been shown to be essentially determined by matrix metalloproteinase 7 (MMP-7), an enzyme, whose activation has been shown to be regulated by myeloperoxidase.7,8
Editorial, see p 11
Meet the First Author, see p 3
Herein, we show that systemic levels of myeloperoxidase are associated with a history of ventricular arrhythmias in patients undergoing elective diagnostic cardiac evaluation, and that myeloperoxidase is causally linked to arrhythmogenic ventricular remodeling in mice. A proarrhythmogenic role for myeloperoxidase was observed in vivo using Mpo−/− mice following either myocardial ischemia/reperfusion (I/R) injury or permanent ischemia (PI), revealing a role for myeloperoxidase in both the enzymatic degradation of Cx43 and the propagation of ventricular fibrosis through induction of fibroblast-to-myofibroblast transdifferentiation, which are both hallmarks of proarrhythmogenic myocardial remodeling.9,10
An expanded Methods section can be found in the Online Data Supplement.
Male, 8- to 12-week-old wild-type (WT) littermates, myeloperoxidase-deficient (Mpo−/−), and CD11b/CD18 integrin–deficient (CD11b−/−) mice were used for this study. Animals were littermates of C57BL/6J background. The strategy for the generation of Mpo−/− mice has been previously reported.11,12 All animal studies were approved by the Universities of Hamburg and Cologne Animal Care and Use Committees and follow ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.
LAD Artery Ligation
In brief, 8- to 12-week-old WT, Mpo−/−, and CD11b−/− mice were subjected to left thoracotomy. An 8/0 polypropylene suture was placed through the myocardium into the anterolateral LV wall. For I/R, the ligation was removed after 30 minutes to allow ≤7 days of reperfusion. For I/R of CD11b−/− mice, the ligation was removed after 40 minutes to allow ≤2 days of reperfusion. For PI, mice were maintained for ≤21 days without reperfusion. Animals which died during instrumentations or which did not properly recover were excluded from analyses.
Right Ventricular Stimulation
After 7 days of reperfusion or 21 days of PI isoflurane anaesthetized WT, Mpo−/− and CD11b−/− mice were subjected to a protocol of right ventricular stimulation.13 Ventricular tachycardia (VT) was defined as a series of repetitive ventricular ectopic beats lasting for >200 ms.
Implantation of ECG Transmitters, ECG Telemetry, and Arrhythmia Provocation
ECGs were recorded in freely moving unrestrained mice. Twenty-four hours after LAD ligation, arrhythmia provocation was performed by double injections of isoproterenol (IP 2 mg/kg) separated by an interval of 30 minutes.14 VT was defined as a series of repetitive ventricular ectopic beats lasting for >200 ms.
In Vivo Electrophysiological Mapping
After 7 days I/R or 21 days PI, the heart was exposed by thoracotomy. A 32-electrode microelectrode array (Multichannel Systems, Reutlingen, Germany) was positioned on the epicardial surface of the LV. Inhomogeneity index, absolute inhomogeneity, variation coefficient, and mean velocity of interelectrode conduction were calculated.15 For pacing studies, the hearts were stimulated with a concentric bipolar electrode (FHC, Bowdoin) with a stimulus rate of 10 Hz.
In Vitro Electrophysiological Mapping
Differentiation and purification of murine induced pluripotent stem cell–derived cardiomyocytes (iPSCMs) is described elsewhere.16 iPSCMs at day 16 of differentiation were cultured on a 120-electrode microarray. After 5 days of settling, cells were treated over night as indicated. Field potentials were recorded (120pMEA; Multichannel Systems), and activation maps were calculated as described above.
Transthoracic echocardiography was performed using the Vevo 2100 System (VisualSonics, Toronto, Canada).
Action Potential Recordings by Sharp Electrode
Short-axis ventricular slices were prepared from hearts subjected to PI for 3 days as described before.17 Intracellular action potential (AP) recordings were performed in ventricular slices with sharp electrodes (20–40 mol/LΩ resistance when filled with 3 mol/L KCl) made of borosilicate glass capillaries (WPI, Sarasota). For inhibitor experiments, BaCl2 (Sigma Aldrich, Germany) or tetrodotoxin (Abcam, ab120054, Germany) was added, as indicated.
Determination of Fibrotic Area
Paraffin sections were stained with picrosirius red following standard protocols. Images were acquired using a DP25 camera (Olympus, Hamburg, Germany) mounted on a BX51 microscope (Olympus). Mean fibrotic area of 12 sections was quantified using Cell A software (Olympus).
Determination of Infarct Size
Hearts were excised, cut into cross-sections, and, for I/R analyses, incubated in 2,3,5-triphenyl-tetrazolium chloride solution because additional Evans Blue perfusion is unfeasible with reopened LAD. For PI, hearts were excised and injected with Evan blue dye via the aorta ascendens. Infarct area was assessed by planimetry using BZ2-Analyser software (Keyence).
Isolation and Treatment of Adult Ventricular Cardiomyocytes
Hearts were excised and mounted on a constant-flow Langendorff circulation (Radnoti Ltd, Ireland) and retrogradely perfused with Liberase TM (Roche) and trypsin (Invitrogen). Cells were collected and incubated with myeloperoxidase (Planta Natural Products) and H2O2 (Sigma) and an inhibitor to MMP-7 (Calbiochem). Cells were lyzed and processed for immunoblotting.
Murine Myeloperoxidase Plasma Levels
Heparin plasma was analyzed for myeloperoxidase using Mouse myeloperoxidase-ELISA (Hycult Biotech).
Staining for Polymorphonuclear Neutrophil Infiltration
Frozen heart sections (4 µm) were stained with rat antimouse neutrophil Ly6G primary antibody according to standard protocol. Images were acquired using a Prosilica GC camera (Allied Vision Technologies) mounted on a Leica DMLB light microscope.
Immunofluorescence Staining for α–Smooth Muscle Actin and Myeloperoxidase
Frozen heart sections (4 µm) were incubated with primary antibody against α–smooth muscle actin (1:200, rabbit IgG) and DDR-2 (discoidin domain-containing receptor 2; 1:50, goat IgG) or myeloperoxidase (1:250, rabbit IgG) after secondary antibody incubation. Nuclei were stained with DAPI.
Fibroblast Isolation, Stimulation, and Analysis
Ventricles of WT mice were digested in Liberase/Tyrode solution, and cells were incubated for 8 hours. For the analysis of phospho-p38 mitogen-activated protein kinase (MAPK), incubation time was reduced to 15 minutes. For the analysis of collagen type I incubation, time was extended to 36 hours. Unless otherwise indicated, fibroblasts were treated with 10 µg/mL catalytically active or inactive myeloperoxidase and 20 µmol/L H2O2 or 10 µmol/L p38 inhibitor SB203580. Fibroblasts were incubated with WT and Mpo−/− leukocytes for 8 hours. Cells were lyzed and processed for immunoblotting.
Immunoblotting for Cx43
Protein blotting was performed with a modified standard protocol using primary antibodies to Cx43 (1:2000) or GAPDH (glyceraldehyde 3-phosphate dehydrogenase; 1:5000). Cardiac HL-1 cells were treated with either PBS (phosphate-buffered saline), myeloperoxidase (10 µg/µL)+H2O2 (80 µmol/L), or pro-MMP-7 (1 µg/mL) for 16 hours.
Immunofluorescence Staining for Cx43
Staining of frozen heart sections was performed with a modified standard protocol detecting Cx43 and N-cadherin. Images were taken with a Retiga 1300 CCD camera mounted on Leica DMLB fluorescence microscope by iVision v4.0. The infarct region was identified by total absence of Cx43 immunoreactivity.
Cx43 and Ion Channel mRNA Analyses
LV heart tissue was collected. RNA was isolated (RNeasy-Kit, Qiagen), and quantitative real-time PCR (Sso Fast Eva Green, BioRad) was performed according to the manufacturer instructions. Target gene mRNA expression was normalized to mRNA expression of GAPDH by ΔΔCT method.
LV tissue was dissected to infarct and peri-infarct tissue using a dissection microscope (Leica). Peri-infarct tissue was analyzed for MMP-7 activity using the SensoLyte 520 MMP-7 Assay Kit (Anaspec) following manufacturer instructions protocol.
Human Myeloperoxidase Plasma Levels
The study was approved by local ethics committees and was conducted in accordance to the Declaration of Helsinki and the guidelines for good clinical practice. All individuals gave written informed consent before inclusion in the study. Serum myeloperoxidase levels were assessed by CardioMPO (Cleveland Heart Laboratory) assay on autoanalyzer according to manufacturer instructions.
Plasma was analyzed for cytokines by using a LEGENDplex Mouse Inflammation Panel (13-plex, Biolegend) according to manufacturer instructions.
Results are expressed as mean±SD. Statistical analysis was performed, unless otherwise indicated, using Kruskal–Wallis test followed by Bonferroni post hoc test, Mann–Whitney U test, or unpaired Student t test as appropriate. Univariate and multivariate logistic regression analyses were used to determine independent predictors of arrhythmias, adjusting for traditional cardiac risk factors. Regression analyses were performed using JMP Pro version 10 (SAS Institute, Cary, NC) and R (3.1.2, Vienna, Austria). All other calculations were performed using SPSS version 23.0. *P<0.05, **P<0.01, ***P<0.001.
Myeloperoxidase Plasma Levels, Myocardial Neutrophil Infiltration, and Inflammatory Response
To characterize a potential mechanistic link between myeloperoxidase and the inducibility of VT, different mouse models of myocardial ischemia were applied. WT and Mpo−/− mice were subjected to ischemia and reperfusion (I/R) and PI on ligation of the ramus interventricularis anterior (LAD). Myeloperoxidase plasma levels were increased >2-fold in WT mice both 7 days after I/R and 5 days after PI compared with sham-operated (sham) animals. After 21 days of PI, myeloperoxidase plasma levels dropped but were still significantly elevated in comparison to sham-operated animals (Figure 1A). Ly6G staining of the infarct and peri-infarct zones revealed a significantly increased infiltration of polymorphonuclear leukocytes in WT animals compared with Mpo−/− animals 7 days after I/R and, to a lesser, not significant extent, 3 days after PI (Figure 1B and 1C). Of note, overall polymorphonuclear leukocytes infiltration was lower after PI than after I/R. Analyses of multiple plasma cytokine levels at multiple time points by flow cytometry revealed no relevant differences between WT and Mpo−/− mice in either model (Online Figure I).
Infarct Size and LV Function
In accordance with previous reports, infarct size after 7 days I/R and 21 days after PI did not differ significantly between WT and Mpo−/− mice. Expectedly, overall infarct size was much larger in hearts subjected to PI when compared with I/R (Figure 1D). In accordance, more pronounced deterioration of systolic LV ejection fraction was observed after PI than after I/R at multiple time points. No significant differences between WT and Mpo−/− animals were noted at day 3 or 7 of I/R (Figure 1E), whereas on PI, ejection fraction was improved in Mpo−/− mice at day 21 (Figure 1F).
Importance of Neutrophils for Myocardial Myeloperoxidase Accumulation
To investigate the role of altered myocardial neutrophil infiltration and myeloperoxidase release on arrhythmia development after infarction, CD11b/CD18 integrin–deficient (CD11b−/−) mice were subjected to I/R. Expectedly, CD11b−/− mice showed a significantly reduced cardiac neutrophil infiltration when compared with WT littermates 6 hours and 2 days after I/R (Figure 1H and 1J). Importantly, despite elevated plasma levels of myeloperoxidase in CD11b−/− mice, which were similar to WT mice after I/R (Figure 1G), myocardial myeloperoxidase levels were significantly lower than in WT mice (Figure 1I and 1K). Neither neutrophils nor intracardiac myeloperoxidase could be detected after sham operation (data not shown). In line with these observations, supplementation of myeloperoxidase via osmotic mini pumps (data not shown) or daily retro-orbital injections for 7 days did not result in myocardial accumulation of myeloperoxidase within the LV (Online Figure IIA, right) despite supraphysiological plasma levels, suggesting that extravasation of neutrophils into the postischemic tissues is of immanent importance to carry myeloperoxidase into the myocardium.
Inducibility of VT by Right Ventricular Stimulation
At day 7 of I/R and day 21 of PI, WT mice exhibited increased vulnerability to VT (representative ECGs in Figure 2A and 2B) when compared with sham-operated animals (WT sham) with respect to the number of VT episodes (Figure 2C) and the duration of VTs (Figure 2D). In comparison, Mpo−/− mice undergoing I/R (Mpo−/− I/R) or permanent LAD ligation (Mpo−/− PI) showed a significantly reduced vulnerability for VT episodes and duration (Figure 2C and 2D). Over a period of 3 months after permanent LAD ligation, no spontaneous deaths occurred, and, accordingly, no difference in mortality between WT and Mpo−/− mice after PI could be demonstrated (Online Figure III).
CD11b−/− mice, which showed reduced neutrophil infiltration and cardiac myeloperoxidase deposition after I/R subjection, were also significantly protected against VT induction as shown by a lower number and length of VT episodes when compared with WT mice (Figure 2E through 2G). Conversely, intravenous myeloperoxidase infusion in Mpo−/− mice, which was not associated with myocardial myeloperoxidase accumulation, did not re-establish VT vulnerability in Mpo−/− mice (Online Figure IIB and IIC).
Spontaneous VT Development
To assess spontaneous VT development, in vivo ECGs of WT and Mpo−/− mice were recorded 24 hours after LAD ligation and additional challenge with isoproterenol. Mpo−/− mice showed a significantly less frequent development of spontaneous VT than WT (representative ECG traces are shown in Figure 2H). VT probability was lower, VT freedom was longer, and VT number and mean time of VT episodes were lower (Figure 2I through 2L).
In Vivo Epicardial Mapping Studies
Epicardial mapping studies in spontaneously beating and stimulated hearts revealed a disruption of conduction homogeneity in WT animals after I/R and PI, whereas in Mpo−/− mice, it was preserved in both models, as ascertained by respective differences in the inhomogeneity index, absolute inhomogeneity, the variation coefficient of local phase delays, and the mean conduction velocity (Figure 3). Of note, no conduction was detectable within the infarct scar tissue in PI animals (data not shown).
Effects of Myeloperoxidase on Expression and Function of Ion Channels
Transcripts of the sodium voltage-gated channel alpha subunit 5 (Nav1.5), the potassium voltage-gated channel subfamily A member 5 (Kv1.5), subfamily Q member 1 (KVLQT1), subfamily J member 2 (Kir2.1), subfamily J member 11 (Kir6.2), the potassium 2 pore domain channel subfamily K member 3 (TASK-1), and the hyperpolarization activated cyclic nucleotide gated potassium channel 2 (HCN2) were detectable within the infarct region 3 days after permanent LAD ligation. Furthermore, the levels of Nav1.5, KV1.5, Kir6.2, TASK-1, and HCN2 mRNA were changed 3 days after LAD ligation when compared with myocardial tissue from healthy mice. Of importance, mRNA levels did not differ between WT and Mpo−/− infarct tissue on PI (Online Table I).
In addition, APs did not differ between WT and Mpo−/− mice as assessed with sharp electrode measurements of living heart tissue slices after 7 days of PI apart from a shortened APD50 in Mpo−/− mice (WT: 13.2±0.3 ms versus Mpo−/−: 9.5±1.4 ms; P≤0.05). This is likely irrelevant given the absence of a significant effect on APD90 (WT: 88.8±6.0 ms versus Mpo−/−: 67.9±7.8 ms) or APD50/90 (WT: 15.3±0.9% versus Mpo−/−: 14.2±1.6%). To further characterize myeloperoxidase effects on ion channel function, increasing concentrations of ion channel blockers were administered to infarcted tissue slices of WT and Mpo−/− animals. Although the potassium channel blocker BaCl218 revealed no differences between both groups, AP amplitude was significantly decreased in hearts from Mpo−/− animals compared with WT animals after incubation with the sodium channel blocker tetrodotoxin19 (WT: 73.5±4.0 mV versus Mpo−/−: 47.6±5.5 mV; P≤0.05; Online Figure IV).
Cx43 in the Infarct and Peri-infarct Zone
Because connexins, which allow for ion exchange between cardiomyocytes, are an integral part of myocardial conduction homogeneity and are regulated in a redox-sensitive fashion, we tested the effect of myeloperoxidase on Cx43 expression and function.
Immunostainings for Cx43 revealed a complete absence of Cx43 immunoreactivity in the infarct zone of WT and Mpo−/− animals (Figure 4A). In the peri-infarct region, a significantly decreased signal for Cx43 was detected in WT I/R mice compared with sham-operated mice. In contrast, no reduction was recognized in Mpo−/− I/R mice in comparison to sham-operated Mpo−/− mice (Figure 4B and 4C). Accordingly, Mpo−/− hearts revealed significantly higher immunoreactivity for Cx43 in the peri-infarct region 3 days and 21 days after PI induction than WT hearts (Figure 4D and 4E). In both models, I/R and PI, ventricular Cx43 mRNA expression levels did not differ between WT and Mpo−/− animals pointing toward a post-translational effect of myeloperoxidase on Cx43 levels (Figure 4F).
MMP-7–Dependent Cx43 Degradation
Studies in isolated adult cardiomyocytes confirmed a decreased content of Cx43 after incubation with myeloperoxidase. 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 after myocardial ischemia but also to be activated by myeloperoxidase-derived hypochlorous acid (Figure 5A).7,8 In addition, activation of MMP-7 was significantly more abundant in the peri-infarct region of WT than in Mpo−/− mice after I/R in vivo (Figure 5B). To further investigate the effect of myeloperoxidase-mediated Cx43 degradation via MMP-7, conduction patterns of a monolayer of iPSCMs were assessed by in vitro mapping analyses. Importantly, these cells are devoid of MMP-7 as assessed by immunoblotting and quantitative real-time analysis (Online Figure V). Spontaneously beating iPSCM showed homogeneous conduction patterns (Figure 5C) under control conditions. Addition of myeloperoxidase/H2O2 or pro-MMP-7 alone had no effect on the observed patterns. However, concomitant treatment with pro-MMP-7/myeloperoxidase/H2O2 resulted in a severe disruption of conduction homogeneity as demonstrated by an increased inhomogeneity index (Figure 5D), an elevated absolute inhomogeneity (Figure 5E), an increased variation coefficient (Figure 5F), and a reduced mean conduction velocity (Figure 5G), indicative of diminished intercellular electric coupling among iPSCM. Conversion of pro-MMP-7 to active MMP-7 by myeloperoxidase-derived HOCl has been described before.8 Accordingly, incubation of the cardiac muscle cell line HL-1, which also lacks MMP-7 protein expression (Online Figure V), with myeloperoxidase/H2O2/pro-MMP-7 resulted in decreased Cx43 levels, whereas single treatment with myeloperoxidase/H2O2 or pro-MMP-7 did not induce Cx43 degradation (Online Figure VI).
Overall LV fibrosis and interstitial ventricular fibrosis (excluding the infarct scar) 7 days after I/R were profoundly lower in Mpo−/− mice than in WT animals, as demonstrated by myocardial picrosirius red stainings for collagen deposition (Figure 6A through 6C; Online Figure VII). Similarly, Mpo−/− hearts exhibited significantly less fibrosis after 21 days of PI, albeit overall LV fibrosis was again more pronounced than in the I/R model (Figure 6D and 6E; Online Figure VII).
To determine myofibroblast accumulation in ventricular tissue, the main collagen-producing cell type in ventricular myocardium under pathological conditions,20 colocalization of the immunoreactivity for the fibroblast marker DDR-2, and the myofibroblast marker α–smooth muscle actin was assessed in cardiac sections 7 days after I/R and 5 days after PI. Indeed, the number of myofibroblasts was significantly lower in the infarct and peri-infarct region of Mpo−/− hearts than in WT after I/R or PI. The total number of myofibroblasts was slightly but not significantly lower in WT I/R hearts than in WT PI hearts (P=0.151; Figure 7A and 7B). Immunoblot analyses revealed dose-dependent transdifferentiation of isolated cardiac fibroblasts to myofibroblasts on incubation with myeloperoxidase in vitro. Strikingly, this equaled the effect of PDGF (platelet-derived growth factor) treatment, an established inducer of fibroblast differentiation (Figure 7C). The incubation of fibroblasts with myeloperoxidase leads to a significantly increased expression of collagen type I compared with untreated cells (Figure 7D). Furthermore, coculture of isolated fibroblasts with isolated WT leukocytes leads to a significantly more pronounced transdifferentiation of fibroblasts when compared with fibroblasts cocultured with leukocytes isolated from Mpo−/− mice (Figure 7E). Next, we tested whether myeloperoxidase induces p38 MAPK phosphorylation in isolated fibroblasts, an inducer of fibroblast-to-myofibroblast transdifferentiation. Indeed, Western blot analyses revealed an augmented amount of p38 MAPK phosphorylation on myeloperoxidase treatment, a process which was completely abolished on additional p38 inhibitor treatment or treatment with catalytically inactive myeloperoxidase (Figure 7F).
Systemic Myeloperoxidase Levels Are Associated With Ventricular Arrhythmias
To test whether myeloperoxidase is linked to the occurrence of arrhythmias in humans, we retrospectively measured circulating levels of myeloperoxidase in a high-risk cohort of stable patients undergoing elective diagnostic cardiac evaluations during coronary angiography and who were no candidates for primary prevention with an implantable cardioverter-defibrillator (ejection fraction >35%).1,21 Table 1 shows the baseline characteristics of the 2622 subjects included in the study cohort. The mean age of the population was 63.1±11.1 years; 76% of patients had coronary artery disease. Patients with arrhythmias were older, had a lower ejection fraction, and more often received angiotensin-converting enzyme inhibitors. Binary logistic regression analysis revealed that myeloperoxidase plasma levels are associated with a history of ventricular arrhythmias, sudden cardiac death, and/or implantable cardioverter–defibrillator implantation (odds ratio, 1.76; 95% CI, 1.19–2.6, highest versus lowest tertile) and remain correlated after adjustment for pertinent risk factors (odds ratio, 1.83; 95% CI, 1.23–2.73, highest versus lowest tertile; Table 2).
Herein, it is shown that myeloperoxidase promotes proarrhythmic remodeling and ventricular arrhythmias after myocardial ischemia. Studies in different animal models of ischemia-related myocardial damage using Mpo−/− mice and studies using spontaneously beating iPSCMs reveal that myeloperoxidase augments arrhythmogenic LV remodeling as manifested in (1) breakdown of Cx43 by activation of MMP-7, and (2) enhanced ventricular fibrosis by transdifferentiation of fibroblasts, which ultimately leads to (3) pronounced electric conduction slowing and disruption of conduction homogeneity, and (4) increased susceptibility to VT.
The mechanisms underlying myocardial damage in I/R and PI are diverse,22 but neutrophils have long been regarded as a crucial component.23,24 The neutrophil-derived enzyme myeloperoxidase has been investigated in the context of ischemic myocardial damage. Vasilyev et al4 showed that myeloperoxidase depletion had no effect on infarct size after myocardial I/R, whereas Askari et al5 demonstrated preserved LV end-diastolic diameter and LV function in Mpo−/− mice. Instigated by these studies, we were able to show that myeloperoxidase also exerts proarrhythmogenic effects in the context of postischemic myocardial remodeling. We identified Cx43 degradation and increased LV fibrosis as underlying mechanisms for this observation. Cx43 is the principal gap junctional protein in the LV, which is critically linked to ventricular homogeneity of electric conduction and ventricular arrhythmias.25 As outlined above, an important mechanism of peri-infarct disruption of gap junctional integrity is MMP-7–dependent degradation of Cx43.7 MMP-7 has previously also been shown to be specifically activated by myeloperoxidase.8 Indeed, we were able to demonstrate reduced peri-infarct MMP-7 activity in Mpo−/− mice and abrogation of myeloperoxidase-dependent Cx43 degradation by inhibition of MMP-7 in isolated adult murine cardiomyocytes. The dependence of myeloperoxidase effects on MMP-7 is strongly underlined by in vitro mapping studies of iPSCMs. These cells do not express MMP-7, and indeed conduction homogeneity was not affected by myeloperoxidase and H2O2 alone but only after addition of myeloperoxidase/H2O2 and pro-MMP-7.
Fibrosis is regarded as a critical substrate for ventricular arrhythmias, and myeloperoxidase profibrotic effects, for example, by oxidative inactivation of plasminogen activator inhibitor-1, have previously been demonstrated.5 We therefore assessed ventricular fibrosis in our animal models and found that Mpo−/− mice were protected. Because activation of MMP-7 is not involved in profibrotic remodeling after infarction,26 we assessed fibroblast-to-myofibroblast transdifferentiation. Myofibroblasts maintain their secretory activity of collagen ≤90 days and are a significant component of progressive adverse cardiac remodeling.20 As evidenced by immunostainings of myocardial sections and functional in vitro studies, we could demonstrate myeloperoxidase-dependent fibroblast transdifferentiation and could link this effect to myeloperoxidase-derived HOCl-dependent p38 MAPK activation,27 thus disclosing a novel, additional profibrotic property of myeloperoxidase.
Ion channel alterations in the peri-infarct region have been demonstrated to be of relevance for ischemia-related cardiac arrhythmias.28 We have previously shown that myeloperoxidase does not affect AP characteristics in isolated cardiomyocytes.6 Because studies on isolated cardiomyocytes only crudely approximate the conditions of cells integrated in the myocardial cell network within the peri-infarct region, we performed sharp electrode measurements on living tissue slices of infarcted murine hearts and found no relevant differences between hearts from WT and Mpo−/− mice. However, Mpo−/− hearts showed an increased susceptibility to voltage-gated sodium channel inhibition by tetrodotoxin as manifested by a decreased AP amplitude. This points toward post-translational modifications of voltage-gated sodium channels by myeloperoxidase, which could be responsible for differences in susceptibility to arrhythmias. Although further investigation of this interesting observation is clearly warranted, the absence of an effect of myeloperoxidase deficiency on APs in untreated tissue slices and the strong effects of myeloperoxidase deficiency on Cx43 and fibrosis together with the reduced epicardial conduction velocity suggest a more dominant role for these latter mechanisms in the context of this study.
It is of great interest for myeloperoxidase mode of action that infiltration of neutrophils seems to be required for accumulation of myeloperoxidase in the ventricular myocardium as suggested by our studies on CD11b−/− mice and myeloperoxidase supplemented Mpo−/− mice. Indeed, this observation could provide an additional explanation for the limited usefulness of myeloperoxidase as a biomarker for myocardial infarction because myeloperoxidase plasma levels might only partially correlate with local abundance of myeloperoxidase.
Certainly, this study bears some limitations. It remains open whether the mechanisms revealed herein will finally affect mortality in human pathophysiology. As shown previously, this animal model is not yielding significant mortality—in fact, we and others did not observe any deaths of mice on 3 months after PI.29,30 Moreover, the complex pathogenesis of ventricular arrhythmias on myocardial ischemia, which is based on electric remodeling of ion channels and calcium kinetics, can only be in part appreciated in a mouse model. However, mouse models have been widely used and have allowed broad insight into the pathogenesis of VTs when focusing on connexin-dependent electric homogeneity and fibrosis.13,31–33 Given the fact that myeloperoxidase is much less expressed in murine polymorphonuclear leukocytes when compared with human neutrophils, the observed effects potentially underestimate the role of myeloperoxidase in this disease in humans. In addition, it clearly has to be assumed that next to the specific effects of myeloperoxidase on Cx43 and fibroblast transdifferentiation, which were evident in the absence of neutrophils in our in vitro experiments, myeloperoxidase promotes perpetuation of local inflammation by its chemoattractant and neutrophil-activating properties.34,35 In addition, other pathways, such as oxidative inactivation of plasminogen activator inhibitor-1, are known to contribute to the observed effects of myeloperoxidase on fibrotic remodeling, as has previously been demonstrated.5 Many limitations also apply to our clinical observational data. Some baseline parameters were not balanced between both groups, which can only partly be corrected by multivariate analysis. In addition, myeloperoxidase levels were only examined in subjects subjected to coronary angiography at 1 point in time at a single tertiary referral center; therefore, we cannot exclude selection bias for patients undergoing diagnostic cardiac catheterization. Given these limitations, the data can only be regarded as a pilot study and should be confirmed in independent cohorts. In addition, whether the prognostic value of myeloperoxidase would be further increased on serial myeloperoxidase assessments remains unknown. Furthermore, the implementation of myeloperoxidase as a biomarker to predict VT seems problematic because of the fact that plasma myeloperoxidase levels will only in part reflect myocardial myeloperoxidase activity. Thus, the clinical usefulness of myeloperoxidase as a biomarker for arrhythmic events cannot be derived from the present data and needs to be further studied. However, the current data set comprises the largest population tested for an inflammatory biomarker indicating ventricular arrhythmogeneity, which in our view confirms the mechanistic data reported herein.
In conclusion, the current data reveal that myeloperoxidase affects electric conduction in the ischemic LV and thereby increases the vulnerability for ventricular arrhythmias. Mechanistically, myeloperoxidase impairs Cx43 integrity through the activation of MMP-7 and induces fibrotic remodeling by stimulating fibroblast transdifferentiation to myofibroblasts through activation of p38 MAPK. 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.
We thank Adnana Paunel-Görgülü for providing the HL-1 cells, Dirk Isbrandt for supporting the telemetry experiments, and Christina Schroth and Katja Urban for expert technical assistance.
Sources of Funding
This work was supported by the Deutsche Forschungsgemeinschaft (KL 2516/1-1 to A. Klinke; BA 1870/7-1, BA 1870/9-1, and BA 1870/10–1 to S. Baldus; AD 492/1-1 to M. Adam; RU 1876/1-1 and RU 1876/3-1 to V. Rudolph); The Ministry of Education, Youth and Sports CR (the National Program of Sustainability II No. LQ1605 to L. Kubala); and the Center for Molecular Medicine Cologne funding (Baldus 2-GA and B-02). The GeneBank studies were supported by National Institutes of Health grants P01HL076491, R01HL126827, and the Cleveland Clinic Clinical Research Unit of the Case Western Reserve University CTSA (UL1TR 000439).
S.L. Hazen reports being named as coinventor on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics and therapeutics; having been paid as a consultant for the following companies: Esperion and P&G; receiving research funds from Abbott, P&G, Pfizer Inc, Roche Diagnostics, and Takeda; and having the right to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics from Cleveland Heart Lab, Siemens, Esperion, and Frantz Biomarkers, LLC. The other authors report no conflicts.
In March 2017, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15.11 days.
↵The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.117.310870/-/DC1.
- Nonstandard Abbreviations and Acronyms
- action potential
- integrin α M
- Connexin 43
- ischemia and reperfusion
- Induced pluripotent stem cell–derived cardiomyocytes
- left anterior descending
- left ventricular
- Mitogen-activated protein kinases
- matrix metalloproteinases
- permanent ischemia
- ventricular tachycardia
- wild type
- Received February 21, 2017.
- Revision received March 20, 2017.
- Accepted April 11, 2017.
- © 2017 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Myeloperoxidase (MPO) is released by infiltrating polymorphonuclear neutrophils and has traditionally been viewed as a microbicidal enzyme.
Accumulating evidence demonstrates involvement of MPO in cardiovascular disease, including atherosclerosis and myocardial disease.
Even with optimal medical therapy according to current standards, life-threatening arrhythmias caused by left ventricular remodeling are a major healthcare concern.
What New Information Does This Article Contribute?
MPO promotes proarrhythmogenic remodeling in different models of myocardial ischemia and increases vulnerability for ventricular tachycardia in this setting.
MPO augments postinfarct connexin 43 degradation via matrix metalloproteinase 7 activation, aggravates fibroblast-to-myofibroblast transdifferentiation via p38 mitogen-activated protein kinase phosphorylation, and increases postinfarct collagen deposition and fibrosis development.
MPO deficiency decreases heterogeneity of electric conduction and reduces conduction block, thereby reducing the development of reentry circuits.
Despite intensive scientific efforts, ischemic heart disease is still a leading cause of morbidity and mortality in western countries. Electric and structural remodeling of the left ventricle after myocardial infarction is the most common substrate for ventricular tachycardia and subsequent sudden cardiac death. In this context, the discovery of new potential therapeutic targets is urgently required. MPO was recently identified as predictor of cardiovascular disease. Herein, we identify MPO as a promoter for ventricular tachycardia vulnerability via postinfarct connexin 43 degradation and fibrosis and subsequent ventricular conduction inhomogeneity. MPO may therefore emerge as a novel therapeutic target for pharmacological antiarrhythmic therapies after myocardial infarction.