Concomitant Activation of OSM and LIF Receptor by a Dual-Specific hlOSM Variant Confers Cardioprotection after Myocardial Infarction in Mice

Figure 1. Species-specific binding differences of LIF and OSM to OSMR and LIFR in human and mice. (A) Schematic illustration of species-specific binding properties of human OSM (hOSM), human LIF (hLIF), murine OSM (mOSM) and murine LIF (mLIF). Note that the generation of a human-like OSM (hlOSM) mutant mimics the binding properties of hOSM with the OSMR and LIFR in mice [17]. (B) Three-dimensional model of mOSM, mLIF and hlOSM. The AB loop and D–helix of each molecule constitute structural determinants of their species-specific receptor binding properties. The hlOSM protein contains the human AB loop sequence, which is highlighted in green.

Figure 2. Transcriptome and gene set enrichment analysis characterizes STAT3, STAT5 and c–Myc as major common signaling molecules downstream of OSMR and LIFR activation in cardiomyocytes. (A) Experimental set-up for transcriptome analysis of primary murine cardiomyocytes after treatment with mLIF, mOSM and hlOSM (each at 20 ng mL−1) for 24 h (n = 3). Addition of equivalent volumes of sterile PBS served as control. (B) Principal component analysis of cultured cardiomyocytes treated with PBS, mLIF, mOSM and hlOSM. (C) Number of differentially up- and downregulated genes in mLIF–, mOSM– and hlOSM–treated versus PBS-treated cardiomyocytes. (D,E) Venn diagram of down- and up-regulated differentially expressed genes (DEG) in mLIF–, mOSM– and hlOSM–treated cardiomyocytes. (F) Gene set enrichment analysis (GSEA) of mLIF–, mOSM– and hlOSM–treated cardiomyocytes. GSEA was performed by pairwise comparisons of all cytokine-treated versus control samples. Gene sets are ranked by a normalized enrichment score.

Figure 3. OSMR- and LIFR-mediated activation of STAT3 but not STAT5 and c–Myc coincides with an increased survival of cultured cardiomyocytes under hypoxic conditions. Representative images and semi-quantitative analysis of immunoblots of (A,B) phosphorylated (p–) STAT3 (Tyr705), (C,D) p–STAT5 (Tyr694) and (E,F) p–c–Myc (Ser62) in cardiomyocytes cultured under normoxic and hypoxic conditions (n = 5). Statistical analysis was performed by one-way ANOVA together with Bonferroni post-test comparisons to assess differences under normoxic and hypoxic conditions. Asterisks indicate Bonferroni post–hoc test significances between cytokine-treated versus PBS-treated cardiomyocytes with ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Data represent ratios (FC) of expression in cytokine-treated versus PBS-treated cardiomyocytes. Total STAT3, STAT5 and c–Myc expression is shown below. (G,H) Representative immunofluorescent images and quantitative analysis of Calceinpos viable cardiomyocytes (green) cultured under normoxic and hypoxic conditions (Normoxia +PBS, n = 2; Hypoxia +PBS, +mLIF, +mOSM and +hlOSM, n = 4). Hoechst 33342 (blue) visualizes nuclei of cardiomyocytes. (I) Statistical analysis of…

Figure 4. Kinetic expression pattern of OSM, LIF and their corresponding receptors OSMR and LIFR in cardiac tissue after the onset of myocardial infarction in mice. (A) Schematic illustration of kinetic expression profiling. Cardiac tissue was harvested at indicated time points post-MI and fractionated into a non-infarcted remote zone (RZ) and infarction zone (IZ). Expression of OSMR, OSM, LIFR and LIF in RZ and IZ was analyzed by immunoblotting (n = 6 at Day 1; n = 5 at Day 2; n = 7 at Day 4; n = 5 at Day 7; n = 5 at Day 14 post-MI). (B) Two representative immunoblots of OSMR, OSM, LIFR and LIF in RZ and IZ at indicated time points post-MI. (C–F) Semi-quantitative analysis of immunoblots shown in (B) based on the adjusted mean volume pixel density of bands. Statistical analysis was performed by two-way ANOVA. Asterisks indicate Bonferroni post-hoc test significances between RZ and IZ at individual time points as well as between RZ and IZ throughout the period of observation with * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Data are presented as mean ± sem. Immunofluorescence analysis of (G) OSMR and (H) LIFR in infarcted hearts of mice 7 days after MI. F-actin: grey. OSMR:…

Figure 5. Post-infarction administration of mLIF, mOSM and hlOSM modulates the activation of STAT3, STAT5 and c–Myc at distinct sites of the myocardium. (A) Schematic illustration of systemic post-infarction administration of mLIF, mOSM and hlOSM in mice during the first three days of cardiac remodeling. At Day 4 post-MI, hearts were harvested, fractionated into a non-infarcted remote zone (RZ), border zone (BZ) and an infarction zone (IZ) to perform spatial expression analysis of signaling molecules via immunoblotting. Two representative images and semi-quantitative analysis of immunoblots of (B,C) p–STAT3 (Tyr705), (D,E) p–STAT5 (Tyr694) and (F,G) p–c–Myc (Ser62) in RZ, BZ and IZ of mice (n = 4 for all groups). Semi-quantitative analysis of immunoblots is based on the adjusted mean volume pixel density of bands. Statistical analysis was performed by one-way ANOVA together with Bonferroni post-test comparisons in order to monitor myocardial site-specific effects upon administration of PBS, mOSM, mLIF and hlOSM. Asterisks indicate Bonferroni post-hoc test significances between cytokine- and PBS-treated mice in RZ, BZ and IZ with * p < 0.05, *** p < 0.001 and **** p < 0.0001. Data are…

Figure 6. Simultaneous activation of the OSMR and LIFR by hlOSM preserves cardiac architecture and contractility after the onset of myocardial infarction. (A) Schematic illustration of the different global myocardial strain directions of the left ventricle: longitudinal shortening, circumferential shortening and radial thickening. Statistical analysis of magnetic resonance imaging-based (B) global longitudinal strain, (C) global circumferential strain and (D) global radial strain of the left ventricle at Day 28 post-MI following systemic injections of 100 ng recombinant mLIF, mOSM and hlOSM per gram of body weight at Days 0, 1, 2 and 3. Injections of equivalent volumes of sterile PBS, i.e., 100 μL per mouse, served as controls (+PBS, n = 8; +mLIF, n = 9, +mOSM, n = 8 and +hlOSM, n = 9). (E) Schematic illustration of the subdivision of the left ventricle into basal, midventricular, and apical segments used for regional circumferential strain (RCS) analyses. Statistical analysis of (F) basal RCS, (G) midventricular RCS and (H) apical RCS. Statistical analysis was performed by one-way ANOVA. Asterisks indicate Bonferroni post-hoc test significances between cytokine- and PBS–treated mice with…