aYAP modRNA reduces cardiac inflammation and hypertrophy in a murine ischemia-reperfusion model

(A) Experimental design. IR and modRNA injection was performed as described in the Materials and Methods section. Red fluorescence beads were injected into the left ventricle cavity after LAD ligation to label blood perfused area. 1 d after IR, EdU and MF20 antibodies were intraperitoneally injected to label CMs proliferation and necrosis, respectively. At day 2 after IR, hearts were collected for histology analysis. (B) MF20 staining of heart cross sections. Bar = 500 µm. (C) Quantification of myocardium with MF20 signals. t test: *P < 0.05, n = 4. (D) Triphenyltetrazolium chloride staining of heart cross sections 2 d after IR. Bar = 200 µm. (E) Quantification of myocardial scar size. t test: *P < 0.05, n = 4. (F) Peripheral blood serum troponin T concentration at different days after IR and aYAP modRNA treatment. Sham n = 3; Veh+IR, n = 4; aYAP+IR. t test: *P < 0.05.

(A) Representative images of hematoxylin and eosinstained heart sections. In Veh+IR and aYAP+IR, typical images from the ischemia regions were shown. Bar = 50 µm. (B) Quantification of non-CMs in the infarct region. t test: *P < 0.05, n = 4. (C) Heart sections stained with Ly6G antibody, low magnification. White dotted lines indicate Ly6G⁺ myocardium. Bar = 500 µm. (D) Quantification of Ly6G⁺ myocardium. Ly6G⁺ myocardium area was normalized against the whole myocardium area. t test: *P < 0.05, n = 4. (E) High magnification of heart sections stained with Ly6G antibody. Images were taken from infarct zone. Bar = 50 µm. (F) Ly6G⁺ cell density in the infarct zone. (A, B, C, D, E, F) Heart sections from day 2 (D2) after IR were used. (G, H, I) Quantification of different myeloid lineage leukocytes. Myocardiums from D2 were dissociated and non-CMs were enriched. For flow cytometry analysis, non-CMs were stained with indicated antibodies. Myeloid lineage leukocytes were labeled with CD45 antibody. CD45⁺ cells were further separated into neutrophils (CD11b⁺; Ly6G⁺) and macrophages/monocytes (CD11b⁺; F4/80⁺). Cell number was normalized to the myocardium weight. One-way ANOVA post hoc tests. N = 4. *P < 0.05. (J, K, L, M) Peripheral blood cell composition at day 1 (D1) and day 2 (D2) after IR. Blood samples from affected mice were collected and analyzed on a HEMAVET 950FS auto blood analyzer. The counts of white blood cells, red blood cells, neutrophils, and lymphocytes were automatically analyzed. k/μl, 1,000 cells per microliter blood. Sham, n = 3. Veh+IR, n = 4; aYAP+IR. One-way ANOVA post hoc tests. *P < 0.05.

(A) Experimental design. IR and modRNA injection was performed as described in the material and methods. Red fluorescence beads were injected into the left ventricle after LAD ligation to label blood perfused ventricle. In this long term study, mouse heart function was measured by echocardiography at 1 d, 1, and 4 wk after IR. 1 mo after IR and modRNA treatment, hearts were collected for analysis. (B) EF% measured by echocardiography at 1 d after IR. Sham, n = 4; Veh+IR, n = 6; aYAP+IR, n = 5. (C) EF% measured by echocardiography. EF% was analyzed by paired t test. (D) Heart weight and tibia length ratio. Sham, n = 6; Veh+IR, n = 10; aYAP+IR, n = 10. One-way ANOVA post hoc tests. *P < 0.05. (E) Upper panel: morphology and AAR of hearts receiving different treatment. Micro red fluorescence beads were used to indicate blow flow. AAR region without red fluorescence beads was depicted by dotted lines. Lower panel: Sirius Red and fast green staining of heart sections. Bar = 1 mm. (F) Ratio between AAR and left ventricle surface area. N = 5. (G) Scar size normalized with AAR. (H) Septum thickness. (F, G, H) N = 5. t test *P < 0.05. (I) WGA staining of heart cross sections. Bar = 50 µm. (J) Quantification of septum CM cross-sectional area. In each group, 150 septum CMs were randomly measured. Mann–Whitney test: **P < 0.01. (K) qRT-PCR measurement of Mhy6 and Nppa mRNA level. N = 4. One-way ANOVA post hoc tests. *P < 0.05.

(A) GSEA. DE (DE) genes from YAP gain-of-function or Tead1 loss-of-function data set were analyzed against an immunology gene list. YAP OE NRVMs with YAP gene overexpressed. Tead1^cKO, heart-specific Tead1 knockout. Immune genes were enriched in the down-regulated genes in YAP OE NRVM and in the up-regulated genes of Tead1^cKO heart. (B) Venn diagram of three different gene list sets: immunology genes, DE genes in the Tead1^cKO data set, and DE genes in the YAP OE NRVM data set. In these three gene list sets, 67 common genes were identified. (C) Signaling pathway GO term analysis. The list of 67 genes identified in (B) was used for GO term signaling pathway enrichment analysis (http://geneontology.org). PANTHER overrepresentation test tool was used to analyze the enriched signaling pathways. Bar graph was plotted based on enrichment score. Gene number enriched in related pathways was labeled on each bar. Statistical significance was shown by false discovery rate value. (D) mRNA of Cd14 and Toll receptors and in Yap^cKO mouse heart. The expression values of different Tlr genes were normalized to Gapdh. RNA was collected from 1 mo old Myh6:Cre; Yap^fl/fl (Yap^cKO) mouse heart. N = 4. *P < 0.05. (E) qRT-PCR measurement of Cd14, Tlr2, and Tlr4 in the NRVMs. NRVMs were treated with indicated virus for 48 h in the absence of serum. N = 4. t test: *P < 0.05. (F, G) mRNA of Cd14 (F) or Tlr genes (G) in 1 mo old Myh6:Cre; Yap^fl/fl (Yap^cKO) mouse hearts. The expression values of different Tlr genes were normalized to GAPDH. N = 4. t test: *P < 0.05.

In the absence of serum, NRVMs were first treated with the indicated virus for 12 h, then 1 µg/ml LPS was added to activate TLR4 signaling. 24 h after LPS stimulation, NRVMs were collected for gene expression analysis or cell death studies. (A) Cell morphology after LPS and indicated adenovirus treatment. Cardiac troponin I (TNNI3) was used to label CMs. Bar = 100 µm. (B) Cell viability assay. Cells were treated with MTS solutions and OD 490 was measured according to manufacturer’s protocol (Promega). N = 6. (C, D) NRVM necrosis analysis. (C) Representative images of PI stained NRVMs. Bar = 100 µm. (D) Quantification of PI-positive CMs. N = 4. (E, F) NRVM apoptosis analysis. (E) CMs stained with Apopxin Green. Bar = 100 µm. (F) Quantification of Apopxin-positive CMs. N = 4. (G, H, I) NRVM gene expression analysis. (G) mRNA level of known YAP target genes. (H) mRNA level of crucial innate immune genes regulated by YAP. (I) mRNA level of selected cytokine/chemokine genes. N = 4. (J, K) NRVM necrosis analysis. (J) Representative images of PI stained NRVMs. Bar = 50 µm. (K) Quantification of PI-positive CMs. N = 4. (L) NRVM gene expression analysis. N = 4. (B, D, F, G, H, I, K, L) One-Way ANOVA Post Hoc Tests. *P < 0.05.