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

Figure S1.. aYAP modRNA transduces multiple myocardial cell types. Related with Fig 1. (A) HPLC purification of aYAP modified RNA. (B) Immunofluorescence staining aYAP modRNA transduced myocardium. Lectin was used to label endothelial cells. In the zoom in panel, white arrows indicate endothelial cells transduced by aYAP modRNA. Bar = 50 µm.

Figure 1.. aYAP modRNA expression in the myocardium. (A) aYAP modRNA–transduced cardiomyocytes. 16 h after aYAP modRNA transfection, NRVMs were fixed for immunofluorescence staining. Bar = 50 µm. (B) YAP mRNA level measured by qRT-PCR. *P < 0.05, n = 3. Hearts were collected 2 d after IR and modRNA injection. (C) Immunoblot to show the expression of YAP protein. aYAP modRNA was fused to a 3×Flag tag. Flag antibody immunoblot showed the expression of aYAP in the aYAP modRNA–treated group but not in the vehicle-treated group. (D) Immunohistochemistry staining of Flag-YAP one day (D1), two days (D2) and 1 mo after IR. Bar = 500 µm. (E, F) Immunofluorescence staining of Flag-YAP. Cardiac troponin I (TNNI3) was used to label cardiomyocytes. (E) Low magnification of vehicle or aYAP-transduced myocardium. Bar = 50 µm. (F) high magnification of aYAP-transduced cardiomyocytes. Bar = 25 µm.

Figure 2.. aYAP modRNA increases CM survival. (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.

Figure S2.. Myocardium AAR, CM DNA synthesis, and apoptosis analysis (related with) Figs 2 and 3. (A) Representative images of hearts with IR surgery. Sham, animals with no LAD ligation; D1, 1 day after IR; D2, 2 days after IR; Veh, vehicle. Red fluorescence beads indicate blood perfused region. AARs were outlined with dotted lines. Bar = 1 mm. (B) Quantification of AAR. N = 4. (C) Heart cross section stained with TNNI3. The myocardium was separated into three parts based on TNNI3 signal intensity: remote zone, border zone, and infarct zone. Bar = 500 μm. (D) TUNEL staining of border zone myocardium. TUNEL and TNNI3 double-positive cells were indicated with white arrowheads. Bar = 50 µm. (E) Quantification of TUNEL positive cardiomyocytes in the border zone. N = 4.

Figure S3.. Histological analysis of hearts after IR. Related with Fig 3. (A) Left: representative images of hematoxylin and eosin–stained heart sections (D1). In Veh+IR and aYAP+IR, typical images from the ischemia regions were shown. Bar = 50 µm. Right: quantification of non-cardiomyocytes in the infarct region. N = 4. One-way ANOVA post hoc Tests. *P < 0.05. (B) Left: heart sections stained with Ly6G antibody 1 day (D1) after IR. Bar = 50 µm. Right: quantification of Ly6G+ cells. (C) Left: heart sections stained with Mac-3 antibody on D1. Bar = 50 µm. Right: quantification of Mac-3+ cells on D1. (D) Left: heart sections stained with Mac-3 antibody on D2. Bar = 50 µm. Right: quantification of Mac-3+ cells on D2. (B, C, D) n = 4. One-way ANOVA post hoc tests. *P < 0.05. (E) Cell gating strategy. After side scatter and forward scatter cell size analysis, single cells were sorted based on the expression of different cell surface markers. Gated regions in each plot were further analyzed in the subsequent plot. (F) Representative gating images of CD45+ leukocytes. (G) Representative gating images of CD11b+ and Ly6G+ double-positive neutrophils. (H) Representative gating images of CD11b+ and…

Figure 3.. aYAP modRNA reduces cardiac inflammation after IR. (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…

Figure 4.. aYAP modRNA improves heart function and suppresses cardiac hypertrophy. (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.…

Figure S4.. Related with Fig 4. (A) Heart rate recorded during echocardiography measurement. The measurements were carried out with awake mice. (B) EF% measured by echocardiography. EF% was measured at 1 and 4 wk after IR, and values were analyzed by paired t test. (C) Heart and body weight ratio at 4 wk after IR. One-way ANOVA post hoc tests. *P < 0.05; **P < 0.01. Sham, n = 6; Veh+IR, n = 10; aYAP+IR, n = 10.

Figure 5.. YAP/TEAD1 regulates innate immune gene expression. (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. Tead1cKO, heart-specific Tead1 knockout. Immune genes were enriched in the down-regulated genes in YAP OE NRVM and in the up-regulated genes of Tead1cKO heart. (B) Venn diagram of three different gene list sets: immunology genes, DE genes in the Tead1cKO 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 YapcKO mouse heart. The expression values of different Tlr genes were normalized to Gapdh. RNA was collected from 1 mo old…

Figure S5.. Related with Figs 5 and 6. (A) Heat map of DE immune genes in YAP OE and Tead1cKO microarray data set. Heat map was generated using the ClustVis website heat map tool (https://biit.cs.ut.ee/clustvis/). (B) Immunoblot of NRVMs treated with LacZ or aYAP adenovirus and LPS, as indicated. GAPDH was used as internal control. (C) Representative images of NRVMs stained with PI. Bar = 50 µm. (D) Quantification of PI positive cardiomyocytes. (E) qRT-PCR measurement of Tlr2 and Tlr4 gene expression. Total RNAs from NRVMs treated with indicated conditions were used. One-way ANOVA Post Hoc Tests. (D, E) *P < 0.05. N = 4. (F) Immunoblot of NRVMs treated with LacZ or Cd14-HA adenovirus. HA tagged CD14 was detected by HA antibody.

Figure 6.. aYAP suppresses TLR4 signaling in vitro. 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…

Figure 7.. aYAP improves adult cardiomyocyte survival in vitro. (A, B, C, D) Dissociated adult mouse cardiomyocytes (CMs) were treated with indicated conditions (see details in Materials and Methods section). PI was used to label necrotic cells. (A, C) Representative images of ACMs. Bar = 50 µm. (B, D) Quantification of adult CM death rate. (B, D) n = 4. One-way ANOVA post hoc tests. *P < 0.05. (E, F, G, H) qRT-PCR measurement of gene expression. Total RNA was collected from adult CMs treated with indicated conditions. The expression of different genes was normalized to GAPDH. Gene expression values were analyzed by One-way ANOVA post hoc tests. *P < 0.05, gene expression values were compared between experimental groups and LacZ control group. &, *P < 0.05, values were compared between LPS+LacZ and LPS+aYAP. #P < 0.05, values were compared between LacZ+H2O2 and aYAP+H2O2. N = 3. (I) Schematic summary of the current study. Blunt ended green lines indicate processes suppressed by aYAP.