Acute myocardial infarction (MI) induces an extensive sterile inflammation, which is dominated in the early phase by invading neutrophils and monocytes/macrophages. The inflammatory response after MI critically affects infarct healing and cardiac remodeling. Therefore, modulation of cardiac inflammation may improve outcome post MI. Insulin-like growth factor 1 (IGF1) treatment reduces infarct size and improves cardiac function after MI via IGF1 receptor mediated signaling in myeloid cells. Our study aimed to investigate the effect of IGF1 on neutrophil phenotype both in vitro and in vivo after MI. We show that IGF1 induces an anti-inflammatory phenotype in bone marrow derived neutrophils. On the molecular and functional level IGF1 treated neutrophils were indistinguishable from those induced by IL4. Surprisingly, insulin, even though it is highly similar to IGF1 did not create anti-inflammatory neutrophils. Notably, the IGF1 effect was independent of the canonical Ras/Raf/ERK or PI3K/AKT pathway, but depended on activation of the JAK2/STAT6 pathway, which was not activated by insulin treatment. Single cell sequencing analysis 3 days after MI also showed that 3 day IGF1 treatment caused a downregulation of pro-inflammatory genes and upstream regulators in most neutrophil and many macrophage cell clusters whereas anti-inflammatory genes and upstream regulators were upregulated. Thus, IGF1 acts like an anti-inflammatory cytokine on myeloid cells in vitro and attenuates the pro-inflammatory phenotype of neutrophils and macrophages in vivo after MI. IGF1 treatment might therefore represent an effective immune modulatory therapy to improve the outcome after MI.

Insulin-like growth factor 1 (IGF1) is an anabolic hormone that controls the growth and metabolism of many cell types. However, IGF1 also mediates cardio-protective effects after acute myocardial infarction (AMI), but the underlying mechanisms and cellular targets are not fully understood. Here we demonstrate that short-term IGF1 treatment for 3 days after AMI improved cardiac function after 1 and 4 weeks. Regional wall motion was improved in ischemic segments, scar size was reduced, and capillary density increased in the infarcted area and the border zone. Unexpectedly, inducible inactivation of the IGF1 receptor (IGF1R) in cardiomyocytes did not attenuate the protective effect of IGF1. Sequential cardiac transcriptomic analysis indicated an altered myeloid cell response in the acute phase after AMI, and, notably, myeloid-cell Igf1r-/- mice lost the protective IGF1 function after I/R. In addition, IGF1 induced an M2-like anti-inflammatory phenotype in bone marrow-derived macrophages and enhanced the number of anti-inflammatory macrophages in heart tissue on day 3 after AMI in vivo. In summary, modulation of the acute inflammatory phase after AMI by IGF1 represents an effective mechanism to preserve cardiac function after I/R.

Insulin‐like growth factor 1 (IGF1) preserves cardiac function after myocardial infarction (MI). The remodeling process after myocardial infarction is influenced by the extend of the initial infarction and regulated by multiple cardiac and non‐cardiac cell types. It is unknown whether the beneficial effect of IGF1 on cardiac function is the consequence of a reduction in early myocardial necrosis due to IGF1 receptor (IGF1R) signalling in cardiomyocytes.Inducible cardiomyocyte specific IGF1R KO and wild type mice (WT) underwent 45 min regional myocardial ischemia followed by 4 weeks of reperfusion. At reperfusion, mice received vehicle (Con) or IGF1 as bolus (40 ng/g, ip) followed by continuous infusion over 3 days using osmotic mini pumps (1 μg/g/d, sc). At baseline, there were no differences between WT and KO animals in left ventricular function (end diastolic (EDV), end systolic volume (ESV), and ejection fraction (EF)). IGF1 treatment improved cardiac function not only in WT mice (EF, week 4: 49±4% (IGF1) vs 36±8% (Con)), but also in KO mice (48±5% (IGF1) vs 35±5% (Con)). To investigate the effect of IGF1 on early ischemia and reperfusion injury, C57Bl6 mice underwent 45 min regional myocardial ischemia followed by 2 hours of reperfusion with and without IGF1 treatment. Infarct size staining showed no effect of IGF1 on infarct size (38±10% (IGF1) vs. 39±11% (Con)). In addition, IGF1 did not affect left ventricular function and myocardial damage in isolated perfused mouse hearts after global ischemia.Exogenous IGF1 treatment preserves cardiac function after myocardial infarction independent of cardiomyocyte specific signaling. The preserved function after IGF1 treatment is not caused by an effect on early ischemia and reperfusion injury indicating that IGF1 modulates the cardiac remodeling process in the subacute phase after MI.Support or Funding InformationFunded by the German Research Foundation (SFB1116/A06)

The mammalian heart expresses predominantly two isoforms of protein kinase B (AKT), namely AKT1 and AKT2. Knockout mice for AKT1 and AKT2 respectively, have demonstrated that AKT1 primarily appears to regulate growth whereas AKT2 is rather involved in regulating metabolism. Here we have analyzed common functions of both AKT isoforms in the heart by generating Tamoxifen(OHTX)‐inducible, cardiac myocyte specific AKT1 and AKT2 single and double KO mice (iCMAKT1/2KO). Inactivation of AKT1 and AKT2 resulted in a maximal loss of both isoforms 5 days after start of OHTX‐treatment without a compensatory upregulation of AKT3. The basal level of Ser9 Phosphorylation of GSK3β was significantly reduced in hearts of double KO only, demonstrating that GSK3β is controlled by AKT1 and AKT2 isoforms in CM. Insulin did not stimulate GSK3β phosphorylation. Furthermore, insulin dependent phosphorylation of classical direct and indirect AKT downstream targets like mTor (Ser2448, Ser2841), p70S6 kinase (Thre 389) or S6 ribosomal protein, among others, was shown to be significantly downregulated in iCMAKT1/2KO heart. Functional analysis by echocardiography revealed a progressive decline of pump function characterized by a drop of ejection fraction from initially 62 to 37 % and 31 % on day 10 and day 21, respectively resulting in cardiac failure on day 24. In contrast, the single iCMAKT1 and iCMAKT2 KO mice showed no cardiac depression. Interestingly, wall thickness declined from day 14 to day 21. Concomitantly, cardiac mass declined to 70 %. Gene expression analysis using Agilent 60K microarrays were performed using hearts from single and double knock out mice. On day 10 after KO induction 1095 genes were differentially expressed (P<0.05) and on day 21 this number increased to 7707 (P<0.01, moderated t‐test, Benjamini‐Hochberg correction). On day 10 upon Txf induction Gdf15 (4.9 × up), a typical stress marker for diseased hearts but not ANP was induced in double knock out hearts. On day 21 this expression increased to Gdf15 (58 × up) and also ANP was induced (17.8 × up). None of these expression alterations coud be detected in Akt1 or Akt2 single knock out heart. Ingenuity Pathway analysis identified particularly metabolic pathways as well as EIF2 signalling to be substantially affected. Conclusion Expression of either AKT1 or AKT2 in cardiac myocytes is sufficient to preserve cardiac function whereas loss of both isoforms results in progessive cardiac atrophy and failure. Support or Funding Information This work was funded by DFG Grant IRTG1902.

ATP and its degradation products play an important role as signaling molecules in the vascular system, and endothelial cells are considered to be an important source of nucleotide release. To investigate the mechanism and physiological significance of endothelial ATP release, we compared different pharmacological stimuli for their ability to evoke ATP release from first passage cultivated human umbilical vein endothelial cells (HUVECs). Agonists known to increase intracellular Ca(2+) levels (A23187, histamine, thrombin) induced a stable, non-lytic ATP release. Since thrombin proved to be the most robust and reproducible stimulus, the molecular mechanism of thrombin-mediated ATP release from HUVECs was further investigated. ATP rapidly increased with thrombin (1 U/ml) and reached a steady-state level after 4 min. Loading the cells with BAPTA-AM to capture intracellular calcium suppressed ATP release. The thrombin-specific, protease-activated receptor 1 (PAR-1)-specific agonist peptide TFLLRN (10 μM) fully mimicked thrombin action on ATP release. To identify the nature of the ATP-permeable pathway, we tested various inhibitors of potential ATP channels for their ability to inhibit the thrombin response. Carbenoxolone, an inhibitor of connexin hemichannels and pannexin channels, as well as Gd(3+) were highly effective in blocking the thrombin-mediated ATP release. Specifically targeting connexin43 (Cx43) and pannexin1 (Panx1) revealed that reducing Panx1 expression significantly reduced ATP release, while downregulating Cx43 was ineffective. Our study demonstrates that thrombin at physiological concentrations is a potent stimulus of endothelial ATP release involving PAR-1 receptor activation and intracellular calcium mobilization. ATP is released by a carbenoxolone- and Gd(3+)- sensitive pathway, most likely involving Panx1 channels.

AIMS Recent studies suggested that human umbilical vein endothelial cells (HUVECs) transdifferentiate into cardiomyocytes and smooth muscle cells in vitro. To test the functional relevance of this observation, we examined the transdifferentiation potential of HUVECs in vivo after intracoronary cell application in Wistar rats. METHODS AND RESULTS SPECT measurements (single photon emission computed tomography) revealed that 18% of (111)In-labelled HUVECs infused by intracoronary delivery stably transplanted to the rat heart. For long-term tracking, HUVECs-expressing enhanced green fluorescent protein (EGFP) were infused. Two days following transplantation, HUVECs were positive for caspase-3. Within 3 days, EGFP was associated with individual cardiomyocytes. No labelling of endothelial and smooth muscle cells was observed. The total number of EGFP-labelled cardiomyocytes accounted for 58% of all initially trapped cells. These EGFP positive cells stained negatively for human mitochondrial proteins, but were positive for rat monocarboxylate transporter-1 protein (MCT-1). Furthermore, EGFP-mRNA was detected in these cells by single-cell RT-PCR (reverse transcription followed by polymerase chain reaction). After 21 days, EGFP positive cells were no longer observed. To investigate the underlying mechanism, we generated in vitro apoptotic bodies from EGFP-labelled HUVECs and found them to contain the genetic information for EGFP. Co-incubation of apoptotic bodies with neonatal rat cardiomyocytes caused cardiomyocytes to express EGFP. CONCLUSION When transplanted into the rat heart by efficient intracoronary delivery, EGFP-expressing HUVECs cause the exclusive but transient labelling of cardiomyocytes. Our in vivo findings suggest that it is not cell fusion and/or transdifferentiation that occurs under these conditions but rather a horizontal gene transfer of the EGFP marker via apoptotic bodies from endothelial cells to cardiomyocytes.