The adipokine Retnla deficiency increases responsiveness to cardiac repair through adiponectin-rich bone marrow cells

Figures

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  Fig. 1. Retnla knockout protects mouse heart from myocardial infarction injury. A Survival rates of the experimental groups; wild type (WT, n = 15) mice and Retnla knockout (KO, n = 17) mice with or without myocardial infarction (MI). WT MI n = 47, KO MI n = 36. B Left ventricular ejection fraction (LVEF) was determined by echocardiography in Retnla KO (n = 19) and WT littermates (n = 18) 14 days after MI. C Heart weight/body weight (HW/BW) ratios of mice were calculated before and at 14 days after MI. (WT; n = 7, KO; n = 7, WT MI; n = 18, KO MI; n = 16). D Cardiomyocyte apoptosis (white arrows) in the peri-infarct area was determined by TUNEL staining. cTnI cardiac troponin I. Scale bar, 200 μm. (WT; n = 7, KO; n = 7). E Inflammation-related mediators were analyzed by using antibody array membranes. F Three days after MI, cardiac macrophages were isolated to compare the cell number and phenotype. Ly6G(−)CD11b(+)F4/80(+)CD11C(−) cells are pro-inflammatory macrophages and Ly6G(−)CD11b(+)F4/80(+)CD206(+) cells are anti-inflammatory macrophages. The number of Ly6G(−)CD11b9(+)F4/80(+) cardiac macrophages was higher in the KO mice (n = 3, three mice per sample each) than in the WT mice (n =…

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  Fig. 2. Characterization of Retnla knockout in unsorted bone marrow cells exhibiting cardiac protective effect. A The number of unsorted bone marrow cells (UBCs) was higher in Retnla knockout (KO) mice regardless of myocardial infarction (MI). B Cell survival and proliferation-related mediators were analyzed in UBCs isolated from WT and Retnla KO mice by western blot. C UBCs were isolated from WT and KO mice 2 days after MI, and mRNA levels of adiponectin (Adipoq), p21, and caveolin-1 (Cav1) were quantified by real-time PCR. D The number of phosphorylated histone H3 (pH3)-positive cells in bone marrow was quantified, and the representative images showed pH3-positive cells (red fluorescence) in the bone marrow. (WT MI 4d; n = 14, KO MI 4d; n = 11, WT MI 7d; n = 23, KO MI 7d; n = 23). Scale bar, 200 μm. E To compare the endogenous angiogenic potential of WT and KO mice, mesenchymal stem cells (MSCs) were used. MSCs were isolated from bone marrow and tube formation assay was performed. Tube length, tubular area, and the number of branching points were analyzed in MSCs from wild type and Retnla KO mice. Scale bar, 500 μm. F UBCs were isolated from WT (n = 12) or KO mice (n = 13), and…

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  Fig. 3. Effects of Retnla protein treatment on cardiomyocytes, cardiac fibroblasts, and macrophages. A Cardiac function was assessed by echocardiography in wild type (WT, n = 13) and Retnla transgenic (TG) mice (n = 20) after myocardial infarction (MI). B Cardiomyocyte apoptosis (white arrows) in the peri-infarct area was determined by TUNEL staining. cTnI cardiac troponin I. (WT; n = 7, KO; n = 8). Scale bar, 200 μm. C, D Phosphorylation of stress-activated ERK and p38 and cell cycle arrest-related proteins p21, and were assessed in neonatal rat cardiomyocytes (CM) or neonatal rat cardiac fibroblasts (cFb) by western blotting. Cells were treated with veh or Retnla (500 ng/mL) for 24 h. E, F Expression of pro-apoptotic Bax and antiapoptotic Bcl2 was determined by western blot analysis in cardiomyocytes and cardiac fibroblasts treated with vehicle (veh) or Retnla (500 ng/mL) protein for 24 h. G The ratios of Bax to Bcl2 were calculated in cardiomyocytes and cardiac fibroblasts and showed significant difference. H, I Proliferation of cardiac fibroblasts were induced with FBS with or without Retnla treatment for 24 h. Proliferating cell markers, phosphorylated histone H3 (pH3) and Ki67,…

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  Fig. 4. Involvement of adiponectin in cardiac protection mediated by unsorted bone marrow cells. A The mRNA level of adiponectin (Adipoq) was assessed in neonatal rat cardiomyocytes treated with vehicle (veh) or Retnla (500 ng/mL) for 24 h. B Adiponectin (APN) protein expression was determined by western blot in neonatal rat cardiomyocytes. C Endogenous APN expression was assessed by immunofluorescence staining in neonatal rat cardiomyocytes treated with veh (n = 12) or Retnla (500 ng/mL, n = 15). Adiponectin-expressing cardiomyocytes were counted and quantified. Scale bars, 200 μm. D Relative gene expression analysis of resistin (Retn), brain natriuretic peptide (BNP), tumor necrosis factor receptor superfamily member 12a (TNFRSF12A), cyclin-dependent kinase inhibitor 3 (CDKN3), cyclin D2 (CCND2), and cyclin E1 (CCNE1) in cardiomyocytes treated with veh or Retnla (500 ng/mL) for 24 h. E, F AdipoRon, adiponectin receptor agonist that binds adiponectin receptor 1 and 2, partially restored the transcriptional levels of adiponectin and p21 in Retnla-treated cardiomyocytes and cardiac fibroblasts. G APN protein expression was determined in the myocardium. Scale bar, 400 μm. H Circulating…

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  Fig. 5. Expressional changes of signaling molecules of apoptosis, inflammation, fibrosis, and tissue repair in the heart tissues. Heart tissues were collected at day 2 (A–C) and 14 (D–F) after MI to analyzed signaling molecules. BCL2-associated agonist of cell death (BAD), BCL2-associated X, apoptosis regulator (BAX), and Fas ligand (FasL) are cell death-related molecules. Collagen type IV, α5 (Col4α5), collagen type V, α3 (Col5α3), and transforming growth factor-β1 (TGF-β1) are fibrosis-related molecules. Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-10, and toll-like receptor 2 (TLR2) are inflammation-related molecules. G–I Circulating levels of interleukin-6 (IL-6), IL-1β, and IL-18 in WT mice (n = 5) and KO mice (n = 5) with or without MI were measured. Data are represented as mean ± SEM. #P < 0.05, ##P < 0.01, ###P < 0.001 (by Student’s t test). J Schematic representation showing the proliferative bone marrow milieu with high adiponectin is associated with improved cardiac repair in infarcted myocardium. The images of bone (https://www.onlinewebfonts.com/icon/559079) and heart (https://www.onlinewebfonts.com/icon/493852) is obtained from clipart library, and used under…