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Secretome of apoptotic peripheral blood cells (APOSEC) confers cytoprotection to cardiomyocytes and inhibits tissue remodelling after acute myocardial infarction: a preclinical study.

ABSTRACT: Heart failure following acute myocardial infarction (AMI) is a major cause of morbidity and mortality. Our previous observation that injection of apoptotic peripheral blood mononuclear cell (PBMC) suspensions was able to restore long-term cardiac function in a rat AMI model prompted us to study the effect of soluble factors derived from apoptotic PBMC on ventricular remodelling after AMI. Cell culture supernatants derived from irradiated apoptotic peripheral blood mononuclear cells (APOSEC) were collected and injected as a single dose intravenously after myocardial infarction in an experimental AMI rat model and in a porcine closed chest reperfused AMI model. Magnetic resonance imaging (MRI) and echocardiography were used to quantitate cardiac function. Analysis of soluble factors present in APOSEC was performed by enzyme-linked immunosorbent assay (ELISA) and activation of signalling cascades in human cardiomyocytes by APOSEC in vitro was studied by immunoblot analysis. Intravenous administration of a single dose of APOSEC resulted in a reduction of scar tissue formation in both AMI models. In the porcine reperfused AMI model, APOSEC led to higher values of ejection fraction (57.0 vs. 40.5%, p < 0.01), a better cardiac output (4.0 vs. 2.4 l/min, p < 0.001) and a reduced extent of infarction size (12.6 vs. 6.9%, p < 0.02) as determined by MRI. Exposure of primary human cardiac myocytes with APOSEC in vitro triggered the activation of pro-survival signalling-cascades (AKT, Erk1/2, CREB, c-Jun), increased anti-apoptotic gene products (Bcl-2, BAG1) and protected them from starvation-induced cell death. Intravenous infusion of culture supernatant of apoptotic PBMC attenuates myocardial remodelling in experimental AMI models. This effect is probably due to the activation of pro-survival signalling cascades in the affected cardiomyocytes.

SUBMITTER: Lichtenauer M

PROVIDER: S-EPMC3228946 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Acute occlusion of a coronary artery results in swift tissue necrosis. Bordering areas of the infarcted myocardium may also experience impaired blood supply and reduced oxygen delivery leading to altered metabolic and mechanical processes. While transcriptional changes in hypoxic cardiomyocytes are well-studied, little is known about the proteins that are actively secreted from these cells. We established a novel secretome analysis of cardiomyocytes by combining stable isotope labeling and click chemistry with subsequent mass spectrometry analysis. Further functional validation experiments included ELISA measurement of human samples, murine LAD ligation and adeno-associated virus (AAV) 9-mediated in vivo overexpression in mice. The presented approach is feasible for the analysis of the secretome of primary cardiomyocytes without serum starvation. 1026 proteins were identified to be secreted within 24 hours, indicating a 5-fold increase in detection compared to former approaches. Among them, a variety of proteins have so far not been explored in the context of cardiovascular pathologies. One of the most strongly upregulated secreted factors upon hypoxia was proprotein convertase subtilisin/kexin type 6 (PCSK6). Validation experiments revealed an increase of PCSK6 on mRNA and protein level in hypoxic cardiomyocytes. PCSK6 expression was elevated in hearts of mice following 3 days of ligation of the left anterior descending artery, a finding confirmed by immunohistochemistry. ELISA measurements in human serum also indicate distinct kinetics for PCSK6 in patients suffering from acute myocardial infarction, with a peak on day 3 post-infarction. Transfer of PCSK6-depleted cardiomyocyte secretome resulted in decreased expression of collagen I and III in fibroblasts compared to control treated cells, and siRNA mediated knockdown of PCSK6 in cardiomyocytes impacted transforming growth factor-β activation and mothers against decapentaplegic homolog 3 (SMAD3) translocation in fibroblasts. An Adeno-associated virus (AAV) 9-mediated, cardiomyocyte-specific overexpression of PCSK6 in mice resulted in increased collagen expression and cardiac fibrosis as well as decreased left ventricular function after myocardial infarction. In conclusion, a novel mass spectrometry-based approach allows the investigation of the secretome of primary cardiomyocytes. Analysis of hypoxia-induced secretion led to the identification of PCSK6 to be crucially involved in cardiac remodeling after acute myocardial infarction. Secretome analysis was performed on neonatal rat ventricular cardiomyocytes (NRVCMs) which were incubated under hypoxic conditions (1.5% O2, 5% CO2, 93.5% N2) for 12 (Hypoxia 0-12h), 24 (Hypoxia 0-24h) and 30 (Hypoxia 24-30h) hours. Furthermore, knockdown (KD) of PCSK6 in vitro mediated by small interfering RNA (siRNA) was performed to investigate changes in the secretome of cardiomyocytes with PCSK6 KD vs. control (control siRNA) during 24 hours of hypoxia (PCSK6 KD 0-24h). Cells were pulse-labeled with AHA (L-azidohomoalanine) and SILAC (stable isotope labeling with amino acids in cell culture) for 12 (Hypoxia 0-12h), 24 (Hypoxia 0-24h/PCSK6 KD 0-24h) and 6 hours (Hypoxia 24-30h). For Hypoxia 0-12h 3 replicates, Hypoxia 0-24h 6 replicates, PCSK6 KD 0-24h 3 Replicates and Hypoxia 24-30h 2 replicates were performed with label-swap.

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Secretome of apoptotic peripheral blood cells (APOSEC) confers cytoprotection to cardiomyocytes and inhibits tissue remodelling after acute myocardial infarction: a preclinical study.

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