Macrophages lacking TSC2 have mTORC1-dependent increased GPNMB and ameliorate ventricular dysfunction/remodeling after ischemia-reperfusion

Figures

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  Fig. 1. Constitutive rapamycin sensitive activation of mTORC1 in MΦTSC2−/− BMDMs favors pro-inflammatory polarization responses in vitro. A, Immunoblot of TSC2 protein expression in BMDMs from MΦTSC2+/+versus MΦTSC2−/−mice (n = 5 per group, unpaired t-test). B, Upper: Immunoblot for P70S6K and 4E-BP1 phosphorylation and total protein in BMDMs from same two models incubated with vehicle or rapamycin. (n = 4 per group); Lower: phospho/total protein. C, Left: Immunoblot of Akt activation from same experiment (n = 4 per group), Right: Summary data; all analyzed by one-way ANOVA, Dunnett multiple correction test. D, Proliferation of BMDMs from both groups with vehicle (Con), LPS/IFN-γ or IL-4 stimulation. Upper: cell numbers (n = 20/group), Lower: cell size (n = 60/group). E, Response of BMDMs isolated from each model to IFN-γ + LPS stimulation ± rapamycin (n = 6/group), F BMDM responses to IL-4 ± rapamycin (n = 6/group). Data and mean ± SEM displayed, data normalized to MΦ-TSC2+/+ results for each analysis, using two-way ANOVA with Šidák multiple comparisons test. *P < 1e-20 vs. MΦTSC2+/+, † P < 1e-10, § P < 0.05 vs. vehicle within genotype. 4E-BP1, eukaryotic translation initiation factor…

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  Fig. 2. MΦTSC2−/− mice are protected against post-I/R cardiac dysfunction. A, Representative echocardiographic images at baseline and 4 weeks post– I/R in MΦTSC2+/+ and MΦTSC2−/− mice. B, Quantification of left ventricular ejection fraction, end-diastolic short axis diameter, stroke diameter (end-diastolic – end-systolic diameter), and percent anterior wall thickening (n = 6 per group). C, Lung wet-to-dry weight ratios (n = 6 per group). D, Masson’s trichrome stained cross section with percent fibrosis area (n = 5 per group, unpaired t-test). E, Gene expression for hypertrophic and matrix remodeling genes, data normalized to MΦTSC2−/− (WT) Sham. A-type natriuretic peptide, Nppa, b-myosin heavy chain, Myh7, collagen type 2a1, Col2a1, metalloproteinase 2, Mmp2 (n = 6/group). All data shown with mean ± SD, panels B, C, and E analyzed with two-way ANOVA with Šidák multiple comparisons test.

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  Fig. 3. Rapamycin prevents protection against cardiac I/R injury in MΦTSC2–/– mice. A, B Example M-mode echocardiograms and summary data at 4 weeks post sham or I/R intervention in MΦTSC2+/+ and MΦTSC2–/– mice treated with either vehicle or rapamycin (n = 5/group). Same parameters and analysis as shown in Fig. 2. C Lung wet-to-dry weight ratio, (n = 5/group). D Representative heart sections and fibrotic area quantitation (n = 7/group, unpaired t-test). E Gene expression of hypertrophy and matrix remodeling genes in same hearts (n = 6/group). Analysis in panels B, C, and E are by two-way ANOVA with Šidák multiple comparison test. All data are shown along with mean ± SD.

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  Fig. 4. Reduced myocardial pro-inflammatory immune cell infiltration 5 days post-I/R in MΦTSC2−/− mice. A Number of CD45+ per mg tissue between MΦTSC2+/+ and MΦTSC2–/– hearts (n = 5/group). B Number of CD64+ macrophages per mg in hearts from both models (n = 5/group). C, Representative flow cytometry density plots for CCR2+/CD64+ macrophages (n = 5/group). D, Number of Ly6C+ monocytes per mg tissue in both groups, (n = 5/group). E Representative flow cytometry analysis of macrophage subsets (n = 5/group). F Ly6G+ (neutrophils), CD8+ and CD4+ (T cells), and CD19+ (B cells) each normalized to mg tissue for both groups (n = 5). Analysis used two-tailed unpaired t-tests. Data are mean ± SEM. CD45, leukocyte common antigen; CCR2, C-C chemokine receptor type 2; CD64, Fc gamma receptor 1; Ly6C, lymphocyte antigen 6 complex locus C; Ly6G, lymphocyte antigen 6 complex locus G; MHCII, major histocompatibility complex class II; SEM, standard error of the mean; TSC2, tuberous sclerosis complex 2.

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  Fig. 5. Enhanced GPNMB protein in MΦTSC2–/– BMDM and post IR-myocardium. A Western blot and quantification show increased GPNMB protein levels in BMDMs isolated from MΦTSC2–/– mice compared to MΦTSC2+/+ controls measured under basal conditions (n = 5 per group, unpaired two-tailed t-test). B GPNMB levels elevated in MΦTSC2–/– BMDMs are significantly reduced after rapamycin treatment (n = 4 per group). C Western blot of cardiac tissue lysates 4 weeks post–I/R shows increased GPNMB expression in MΦTSC2–/– hearts that is absent in the cohort treated with rapamycin (n = 4 per group, D ELISA quantification of GPNMB protein from the same cardiac tissues confirmed the elevation in MΦTSC2–/– hearts and its suppression by rapamycin (n = 6 per group). Analysis in panels B–D, one-way ANOVA with Dunnett multiple comparisons correction. Data are mean ± SD. Rapa – rapamycin.