Myocardial infarction (MI) causes a massive increase of macrophages in the heart, which serve various non-redundant functions for cardiac repair. The identities of signals controlling recruitment of functionally distinct cardiac macrophages to sites of injury are only partially known. Previous work identified Regenerating islet-derived protein 3 beta (Reg3β) as a novel factor directing macrophages to sites of myocardial injury. Herein, we aim to characterize functionally distinct macrophage subsets and understand the impact of different members of the Reg protein family including Reg3β, Reg3γ, and Reg4 on their accumulation in the infarcted heart. We have determined dynamic changes of three phenotypically distinct tissue macrophage subpopulations in the mouse heart after MI by flow cytometry. RNA sequencing and bioinformatics analysis identified inflammatory gene expression patterns in MHC-II^hi/Ly6C^lo and MHC-II^lo/Ly6C^lo cardiac tissue macrophages while Ly6C^hi cardiac tissue macrophages are characterized by gene activities associated with healing and revascularization of damaged tissue. Loss- and gain-of-function experiments revealed specific roles of Reg proteins for recruitment of cardiac tissue macrophage subpopulations to the site of myocardial injury. We found that expression of Reg3β, Reg3γ, and Reg4 is strongly increased after MI in mouse and human hearts with Reg3β providing the lead, followed by Reg3γ and Reg4. Inactivation of the Reg3β gene prevented the increase of all types of cardiac tissue macrophages shortly after MI whereas local delivery of Reg3β, Reg3γ, and Reg4 selectively stimulated recruitment of MHC-II^hi/Ly6C^lo and MHC-II^lo/Ly6C^lo but repressed accumulation of Ly6C^hi cardiac tissue macrophages. We conclude that distinct cardiac macrophage subpopulations are characterized by substantially different gene expression patterns reflecting their pathophysiological role after MI. We argue that sequential, local production of Reg proteins orchestrates accumulation of macrophage subsets, which seem to act in a parallel or partially overlapping rather than in a successive manner.

The pleiotropic interleukin-6 (IL-6)–type cytokine oncostatin M (OSM) signals in multiple cell types, affecting processes such as cell differentiation, hematopoiesis and inflammation. In humans, OSM exerts its effects through activation of either of two different heterodimeric receptor complexes, formed by glycoprotein 130 (gp130) and either OSM receptor (OSMR) or leukemia inhibitory factor receptor (LIFR). In contrast, the mouse OSM orthologue acts mainly through dimers containing OSMR and gp130 and shows limited activity through mouse LIFR. Despite their structural similarity, neither human nor mouse OSM signal through the other species' OSMR. The molecular basis for such species-specific signaling, however, remains poorly understood. In order to identify key molecular features of OSM that determine receptor activation in humans and mice, we generated chimeric mouse-human cytokines. Replacing regions within binding site III of murine OSM with the human equivalents showed that the cytokine's AB loop was critical for receptor selection. Substitutions of individual amino acids within this region demonstrated that residues Asn-37, Thr-40 and Asp-42 of the murine cytokine were responsible for limited LIFR activation and absence of human OSMR/LIFR signaling. In human OSM, Lys-44 appeared to be the main residue preventing mouse OSMR activation. Our data reveal that individual amino acids within the AB loop of OSM determine species-specific activities. These mutations might reflect a key step in the evolutionary process of this cytokine, in which receptor promiscuity gives way to ligand-receptor specialization.

Oncostatin M (OSM) and leukemia inhibitory factor (LIF) are closely related members of the interleukin-6 (IL-6) cytokine family. Both cytokines share a common origin and structure, and both interact through a specific region, termed binding site III, to activate a dimeric receptor complex formed by glycoprotein 130 (gp130) and LIF receptor (LIFR) in humans. However, only OSM activates the OSM receptor (OSMR)/gp130 complex. The molecular features that enable OSM to specifically activate the OSMR are currently unknown.To define specific sequence motifs within OSM that are critical for initiating signaling via OSMR, here we generated chimeric OSM−LIF cytokines and performed alanine-scanning experiments. Replacement of the OSM AB loop within OSM's binding site III with that of LIF abrogated OSMR activation, measured as STAT3 phosphorylation at Tyr-705, but did not compromise LIFR activation. Correspondingly, substitution of the AB loop and D-helix in LIF with their OSM counterparts was sufficient for OSMR activation. The alanine-scanning experiments revealed that residues Tyr-34, Gln-38, Gly-39 and Leu-45 (in the AB loop) and Pro-153 (in the D-helix) had specific roles in activating OSMR but not LIFR signaling, while Leu-40 and Cys-49 (in the AB loop), and Phe-160 and Lys-163 (in the D-helix) were required for activation of both receptors. Since most of the key amino acid residues identified here are conserved between LIF and OSM, we concluded that comparatively minor differences in a few amino acid residues within binding site III account for the differential biological effects of OSM and LIF.

Oncostatin M (OSM) and leukemia inhibitory factor (LIF) signaling protects the heart after myocardial infarction (MI). In mice, oncostatin M receptor (OSMR) and leukemia inhibitory factor receptor (LIFR) are selectively activated by the respective cognate ligands while OSM activates both the OSMR and LIFR in humans, which prevents efficient translation of mouse data into potential clinical applications. We used an engineered human-like OSM (hlOSM) protein, capable to signal via both OSMR and LIFR, to evaluate beneficial effects on cardiomyocytes and hearts after MI in comparison to selective stimulation of either LIFR or OSMR. Cell viability assays, transcriptome and immunoblot analysis revealed increased survival of hypoxic cardiomyocytes by mLIF, mOSM and hlOSM stimulation, associated with increased activation of STAT3. Kinetic expression profiling of infarcted hearts further specified a transient increase of OSM and LIF during the early inflammatory phase of cardiac remodeling. A post-infarction delivery of hlOSM but not mOSM or mLIF within this time period combined with cardiac magnetic resonance imaging-based strain analysis uncovered a global cardioprotective effect on infarcted hearts. Our data conclusively suggest that a simultaneous and rapid activation of OSMR and LIFR after MI offers a therapeutic opportunity to preserve functional and structural integrity of the infarcted heart.

It is now accepted that heart failure (HF) is a complex multifunctional disease rather than simply a hemodynamic dysfunction. Despite its complexity, stressed cardiomyocytes often follow conserved patterns of structural remodelling in order to adapt, survive, and regenerate. When cardiac adaptations cannot cope with mechanical, ischemic, and metabolic loads efficiently or become chronically activated, as, for example, after infection, then the ongoing structural remodelling and dedifferentiation often lead to compromised pump function and patient death. It is, therefore, of major importance to understand key events in the progression from a compensatory left ventricular (LV) systolic dysfunction to a decompensatory LV systolic dysfunction and HF. To achieve this, various animal models in combination with an “omics” toolbox can be used. These approaches will ultimately lead to the identification of an arsenal of biomarkers and therapeutic targets which have the potential to shape the medicine of the future.