Mortimer Korf-Klingebiel

GDF-15 is an inhibitor of leukocyte integrin activation required for survival after myocardial infarction in mice

Inflammatory cell recruitment after myocardial infarction needs to be tightly controlled to permit infarct healing while avoiding fatal complications such as cardiac rupture. Growth differentiation factor-15 (GDF-15), a transforming growth factor-β (TGF-β)–related cytokine, is induced in the infarcted heart of mice and humans. We show that coronary artery ligation in Gdf15-deficient mice led to enhanced recruitment of polymorphonuclear leukocytes (PMNs) into the infarcted myocardium and an increased incidence of cardiac rupture. Conversely, infusion of recombinant GDF-15 repressed PMN recruitment after myocardial infarction. In vitro, GDF-15 inhibited PMN adhesion, arrest under flow and transendothelial migration. Mechanistically, GDF-15 counteracted chemokine-triggered conformational activation and clustering of β2 integrins on PMNs by activating the small GTPase Cdc42 and inhibiting activation of the small GTPase Rap1. Intravital microscopy in vivo in Gdf15-deficient mice showed that Gdf-15 is required to prevent excessive chemokine-activated leukocyte arrest on the endothelium. Genetic ablation of β2 integrins in myeloid cells rescued the mortality of Gdf15-deficient mice after myocardial infarction. To our knowledge, GDF-15 is the first cytokine identified as an inhibitor of PMN recruitment by direct interference with chemokine signaling and integrin activation. Loss of this anti-inflammatory mechanism leads to fatal cardiac rupture after myocardial infarction.

Bone marrow cells are a rich source of growth factors and cytokines: implications for cell therapy trials after myocardial infarction

AIMS Results from clinical trials suggest that cardiac function after acute myocardial infarction (AMI) can be enhanced by an intracoronary infusion of autologous unselected nucleated bone marrow cells (BMCs). Release of paracrine factors has been proposed as a mechanism for these therapeutic effects; however, this hypothesis has not been tested in humans. METHODS AND RESULTS BMCs and peripheral blood leucocytes (PBLs) were obtained from 15 patients with AMI and cultured in serum-free medium to obtain conditioned supernatants (SN). BMC-SN stimulated human coronary artery endothelial cell proliferation, migration, and tube formation, and induced cell sprouting in a mouse aortic ring assay. Moreover, BMC-SN protected rat cardiomyocytes from cell death induced by simulated ischaemia or ischaemia followed by reperfusion. While PBL-SN promoted similar effects on endothelial cells and cardiomyocytes, BMC-SN and PBL-SN in combination promoted synergistic effects. As shown by ProteinChip and GeneChip array analyses (each performed in triplicate), BMCs and PBLs expressed distinct patterns of pro-angiogenic and cytoprotective secreted factors. CONCLUSION Our data support the paracrine hypothesis and suggest that characterization of the BMC secretome may lead to an identification of factors with therapeutic potential after AMI.

Myeloid-derived growth factor (C19orf10) mediates cardiac repair following myocardial infarction

Paracrine-acting proteins are emerging as a central mechanism by which bone marrow cell–based therapies improve tissue repair and heart function after myocardial infarction (MI). We carried out a bioinformatic secretome analysis in bone marrow cells from patients with acute MI to identify novel secreted proteins with therapeutic potential. Functional screens revealed a secreted protein encoded by an open reading frame on chromosome 19 (C19orf10) that promotes cardiac myocyte survival and angiogenesis. We show that bone marrow–derived monocytes and macrophages produce this protein endogenously to protect and repair the heart after MI, and we named it myeloid-derived growth factor (MYDGF). Whereas Mydgf-deficient mice develop larger infarct scars and more severe contractile dysfunction compared to wild-type mice, treatment with recombinant Mydgf reduces scar size and contractile dysfunction after MI. This study is the first to assign a biological function to MYDGF, and it may serve as a prototypical example for the development of protein-based therapies for ischemic tissue repair.

Conditional Transgenic Expression of Fibroblast Growth Factor 9 in the Adult Mouse Heart Reduces Heart Failure Mortality After Myocardial Infarction

Background— Fibroblast growth factor 9 (FGF9) is secreted from bone marrow cells, which have been shown to improve systolic function after myocardial infarction (MI) in a clinical trial. FGF9 promotes cardiac vascularization during embryonic development but is only weakly expressed in the adult heart. Methods and Results— We used a tetracycline-responsive binary transgene system based on the &agr;-myosin heavy chain promoter to test whether conditional expression of FGF9 in the adult myocardium supports adaptation after MI. In sham-operated mice, transgenic FGF9 stimulated left ventricular hypertrophy with microvessel expansion and preserved systolic and diastolic function. After coronary artery ligation, transgenic FGF9 enhanced hypertrophy of the noninfarcted left ventricular myocardium with increased microvessel density, reduced interstitial fibrosis, attenuated fetal gene expression, and improved systolic function. Heart failure mortality after MI was markedly reduced by transgenic FGF9, whereas rupture rates were not affected. Adenoviral FGF9 gene transfer after MI similarly promoted left ventricular hypertrophy with improved systolic function and reduced heart failure mortality. Mechanistically, FGF9 stimulated proliferation and network formation of endothelial cells but induced no direct hypertrophic effects in neonatal or adult rat cardiomyocytes in vitro. FGF9-stimulated endothelial cell supernatants, however, induced cardiomyocyte hypertrophy via paracrine release of bone morphogenetic protein 6. In accord with this observation, expression of bone morphogenetic protein 6 and phosphorylation of its downstream targets SMAD1/5 were increased in the myocardium of FGF9 transgenic mice. Conclusions— Conditional expression of FGF9 promotes myocardial vascularization and hypertrophy with enhanced systolic function and reduced heart failure mortality after MI. These observations suggest a previously unrecognized therapeutic potential for FGF9 after MI.

Iron-regulatory proteins secure iron availability in cardiomyocytes to prevent heart failure

Aims Iron deficiency (ID) is associated with adverse outcomes in heart failure (HF) but the underlying mechanisms are incompletely understood. Intracellular iron availability is secured by two mRNA-binding iron-regulatory proteins (IRPs), IRP1 and IRP2. We generated mice with a cardiomyocyte-targeted deletion of Irp1 and Irp2 to explore the functional implications of ID in the heart independent of systemic ID and anaemia. Methods and results Iron content in cardiomyocytes was reduced in Irp-targeted mice. The animals were not anaemic and did not show a phenotype under baseline conditions. Irp-targeted mice, however, were unable to increase left ventricular (LV) systolic function in response to an acute dobutamine challenge. After myocardial infarction, Irp-targeted mice developed more severe LV dysfunction with increased HF mortality. Mechanistically, the activity of the iron-sulphur cluster-containing complex I of the mitochondrial electron transport chain was reduced in left ventricles from Irp-targeted mice. As demonstrated by extracellular flux analysis in vitro, mitochondrial respiration was preserved at baseline but failed to increase in response to dobutamine in Irp-targeted cardiomyocytes. As shown by 31P-magnetic resonance spectroscopy in vivo, LV phosphocreatine/ATP ratio declined during dobutamine stress in Irp-targeted mice but remained stable in control mice. Intravenous injection of ferric carboxymaltose replenished cardiac iron stores, restored mitochondrial respiratory capacity and inotropic reserve, and attenuated adverse remodelling after myocardial infarction in Irp-targeted mice but not in control mice. As shown by electrophoretic mobility shift assays, IRP activity was significantly reduced in LV tissue samples from patients with advanced HF and reduced LV tissue iron content. Conclusions ID in cardiomyocytes impairs mitochondrial respiration and adaptation to acute and chronic increases in workload. Iron supplementation restores cardiac energy reserve and function in iron-deficient hearts.

Identification of Follistatin-Like 1 by Expression Cloning as an Activator of the Growth Differentiation Factor 15 Gene and a Prognostic Biomarker in Acute Coronary Syndrome

BACKGROUND Growth differentiation factor 15 (GDF15) is a stress-responsive cytokine and biomarker that is produced after myocardial infarction and that is related to prognosis in acute coronary syndrome (ACS). We hypothesized that secreted proteins that activate GDF15 production may represent new ACS biomarkers. METHODS We expressed clones from an infarcted mouse heart cDNA library in COS1 cells and assayed for activation of a luciferase reporter gene controlled by a 642-bp fragment of the mouse growth differentiation factor 15 (GDF15) gene promoter. We measured the circulating concentrations of follistatin-like 1 (FSTL1) and GDF15 in 1369 patients with ACS. RESULTS One cDNA clone that activated the GDF15 promoter-luciferase reporter encoded the secreted protein FSTL1. Treatment with FSTL1 activated GDF15 production in cultured cardiomyocytes. Transgenic production of FSTL1 stimulated GDF15 production in the murine heart, whereas cardiomyocyte-selective deletion of FSTL1 decreased production of GDF15 in cardiomyocytes, indicating that FSTL1 is sufficient and required for GDF15 production. In ACS, FSTL1 emerged as the strongest independent correlate of GDF15 (partial R(2) = 0.26). A total of 106 patients died of a cardiovascular cause during a median follow-up of 252 days. Patients with an FSTL1 concentration in the top quartile had a 3.7-fold higher risk of cardiovascular death compared with patients in the first 3 quartiles (P < 0.001). FSTL1 remained associated with cardiovascular death after adjustment for clinical, angiographic, and biochemical variables. CONCLUSIONS Our study is the first to use expression cloning for biomarker discovery upstream of a gene of interest and to identify FSTL1 as an independent prognostic biomarker in ACS.

Incremental Prognostic Value of Biomarkers beyond the GRACE (Global Registry of Acute Coronary Events) Score and High-Sensitivity Cardiac Troponin T in Non-ST-Elevation Acute Coronary Syndrome

BACKGROUND Guidelines recommend the use of validated risk scores and a high-sensitivity cardiac troponin assay for risk assessment in non-ST-elevation acute coronary syndrome (NSTE-ACS). The incremental prognostic value of biomarkers in this context is unknown. METHODS We calculated the Global Registry of Acute Coronary Events (GRACE) score and measured the circulating concentrations of high-sensitivity cardiac troponin T (hs-cTnT) and 8 selected cardiac biomarkers on admission in 1146 patients with NSTE-ACS. We used an hs-cTnT threshold at the 99th percentile of a reference population to define increased cardiac marker in the score. The magnitude of the increase in model performance when individual biomarkers were added to GRACE was assessed by the change (Δ) in the area under the receiver-operating characteristic curve (AUC), integrated discrimination improvement (IDI), and category-free net reclassification improvement [NRI(>0)]. RESULTS Seventy-eight patients reached the combined end point of 6-month all-cause mortality or nonfatal myocardial infarction. The GRACE score alone had an AUC of 0.749. All biomarkers were associated with the risk of the combined end point and offered statistically significant improvement in model performance when added to GRACE (likelihood ratio test P ≤ 0.015). Growth differentiation factor 15 [ΔAUC 0.039, IDI 0.049, NRI(>0) 0.554] and N-terminal pro-B-type natriuretic peptide [ΔAUC 0.024, IDI 0.027, NRI(>0) 0.438] emerged as the 2 most promising biomarkers. Improvements in model performance upon addition of a second biomarker were small in magnitude. CONCLUSIONS Biomarkers can add prognostic information to the GRACE score even in the current era of high-sensitivity cardiac troponin assays. The incremental information offered by individual biomarkers varies considerably, however.

Intracoronary autologous bone marrow cell transfer after myocardial infarction: the BOOST-2 randomised placebo-controlled clinical trial

Aims Intracoronary infusion of autologous nucleated bone marrow cells (BMCs) enhanced the recovery of left ventricular ejection fraction (LVEF) after ST-segment elevation myocardial infarction (STEMI) in the randomised-controlled, open-label BOOST trial. We reassessed the therapeutic potential of nucleated BMCs in the randomised placebo-controlled, double-blind BOOST-2 trial conducted in 10 centres in Germany and Norway. Methods and results Using a multiple arm design, we investigated the dose-response relationship and explored whether γ-irradiation which eliminates the clonogenic potential of stem and progenitor cells has an impact on BMC efficacy. Between 9 March 2006 and 16 July 2013, 153 patients with large STEMI were randomly assigned to receive a single intracoronary infusion of placebo (control group), high-dose (hi)BMCs, low-dose (lo)BMCs, irradiated hiBMCs, or irradiated loBMCs 8.1 ± 2.6 days after percutaneous coronary intervention (PCI) in addition to guideline-recommended medical treatment. Change in LVEF from baseline (before cell infusion) to 6 months as determined by MRI was the primary endpoint. The trial is registered at Current Controlled Trials (ISRCTN17457407). Baseline LVEF was 45.0 ± 8.5% in the overall population. At 6 months, LVEF had increased by 3.3 percentage points in the control group and 4.3 percentage points in the hiBMC group. The estimated treatment effect was 1.0 percentage points (95% confidence interval, -2.6 to 4.7; P = 0.57). The treatment effect of loBMCs was 0.5 percentage points (-3.0 to 4.1; P = 0.76). Likewise, irradiated BMCs did not have significant treatment effects. BMC transfer was safe and not associated with adverse clinical events. Conclusion The BOOST-2 trial does not support the use of nucleated BMCs in patients with STEMI and moderately reduced LVEF treated according to current standards of early PCI and drug therapy.

Effective Blockage of Both the Extrinsic and Intrinsic Pathways of Apoptosis in Mice by TAT-crmA

Evidence accumulates that in clinically relevant cell death, both the intrinsic and extrinsic apoptotic pathway synergistically contribute to organ failure. In search for an inhibitor of apoptosis that provides effective blockage of these pathways, we analyzed viral proteins that evolved to protect the infected host cells. In particular, the cowpox virus protein crmA has been demonstrated to be capable of blocking key caspases of both pro-apoptotic pathways. To deliver crmA into eukaryotic cells, we fused the TAT protein transduction domain of HIV to the N terminus of crmA. In vitro, the TAT-crmA fusion protein was efficiently translocated into target cells and inhibited apoptosis mediated through caspase-8, caspase-9, and caspase-3 after stimulation with α-Fas, etoposide, doxorubicin, or staurosporine. The extrinsic apoptotic pathway was investigated following α-Fas stimulation. In vivo 90% of TAT-crmA-treated animals survived an otherwise lethal dose of α-Fas and showed protection from Fas-induced organ failure. To examine the intrinsic apoptotic pathway, we investigated the survival of mice treated with an otherwise lethal dose of doxorubicin. Whereas all control mice died within 31 days, 40% of mice that concomitantly received intraperitoneal injections of TAT-crmA survived. To test the ability to comprehensively block both the intrinsic and extrinsic apoptotic pathway in a clinically relevant setting, we employed a murine cardiac ischemia-reperfusion model. TAT-crmA reduced infarction size by 40% and preserved left ventricular function. In summary, these results provide a proof of principle for the inhibition of apoptosis with TAT-crmA, which might provide a new treatment option for ischemia-reperfusion injuries.

Corrigendum: Myeloid-derived growth factor (C19orf10) mediates cardiac repair following myocardial infarction

Mortimer Korf-Klingebiel, Marc R Reboll, Stefanie Klede, Torben Brod, Andreas Pich, Felix Polten, L Christian Napp, Johann Bauersachs, Arnold Ganser, Eva Brinkmann, Ines Reimann, Tibor Kempf, Hans W Niessen, Jacques Mizrahi, Hans-Joachim Schönfeld, Antonio Iglesias, Maria Bobadilla, Yong Wang & Kai C Wollert Nat. Med. 21, 140–149 (2015); published online 12 January 2015; corrected after print 19 November 2015