Effects of Cardiomyocyte-Specific Deletion of STAT3–A Murine Model of Heart Failure With Preserved Ejection Fraction

Figure 1. The stat3 gene was actually knock out in STATcKO mice. (A) Experimental design. STAT3flox/flox Cre+ mice were administrated by tamoxifen at 8 weeks and analysis followed indicators at 16 weeks. (B) Representative mouse heart sections immunostained for α-ACTININ (red), STAT3 (green) and DAPI (blue) (C) Western blot analysis showed the levels of STAT3 in WT mice hearts, livers, kidneys and gastrocnemius (n = 3) and STAT3cKO mice hearts, livers, kidneys and gastrocnemius (n = 3). Compared with WT group, *P < 0.05 by Student's unpaired t-test. **P < 0.01 by Student's unpaired t-test.

Figure 2. Cardiomyocyte-specific STAT3 ablation promotes cardiac fibrosis and reduces capillary density. (A) Western blot analysis showed the levels of collagen 1, collagen 3 and fibronectin in WT mice hearts (n = 3) and STAT3cKO mice hearts (n = 3). (B) Representative images of immunohistochemical staining of COL1, COL3, FBN, and CD31 in cardiac left ventricular tissue of WT mice and STAT3cKO mice. (Left panel of every image is original magnification. Right panel of every image is original magnification x10.) (C) Fixed quantity of immunohistochemical staining of COL1, COL3, FBN, and CD31 in cardiac left ventricular tissue of WT mice and STAT3cKO mice (n = 3). Compared with WT group, *P < 0.05 by Student's unpaired t-test. **P < 0.01 by Student's unpaired t-test.

Figure 3. Cardiomyocyte-specific STAT3 ablation induces cardiac hypertrophy without affecting blood pressure. (A) Representative pictures of hearts from a WT mouse and STAT3cKO mouse. (B) Representative images of HE staining in cardiac left ventricular tissue of WT mice and STAT3cKO mice. (C–F) Heart weight (HW), ratio of heart weight to tibia length (HW/TL), ratio of heart weight to body weight (HW/BW), body weight in WT mice (n = 8) and STAT3cKO mice (n = 8). (G) Cardiomyocyte size as assessed by WGA staining in WT mice and STAT3cKO mice. (H) Blood pressure of WT mice (n = 8) and STAT3cKO mice (n = 8). (I–L) Levels of IVS;s, IVS;d, LVPW;s and LVPW;d in echocardiographic observations of WT mice (n = 10) and STAT3cKO mice (n = 10). Compared with WT group, *P < 0.05 by Student's unpaired t-test. **P < 0.01 by Student's unpaired t-test.

Figure 4. Cardiomyocyte-specific STAT3 ablation impaired cardiac diastolic function. (A) The levels of BNP concentration in WT mice (n = 8) and STAT3cKO mice (n = 8). (B) The levels of ST2 concentration in WT mice (n = 8) and STAT3cKO mice (n = 8). (C,D) Representative pictures of pressure-volume loop from a WT mouse and STAT3cKO mouse. (E) Representative left ventricular M-mode echocardiographic tracings of WT mice (n = 10) and STATcKO mice (n = 10). (F,G) Percentages of LVEF and FS in WT mice (n = 10) and STAT3cKO mice (n = 10). (H–K) Levels of E peak, A peak, E/A, E/E' in WT mice (n = 10) and STAT3cKO mice (n = 10). Compared with WT group, *P < 0.05 by Student's unpaired t-test. **P < 0.01 by Student's unpaired t-test.

Figure 5. Cardiomyocyte-specific deletion of Stat3 reduced myocardial PKG levels and RNA-sequencing of STAT3cKO mice hearts suggested potential pathogenesis of HFpEF. (A) The hierarchical clustering method was used to identify the consistency of dysregulated mRNAs in STAT3cKO (n = 3) and WT (n = 3) mice hearts. (B) The colcano map showed the 223 up-regulated gens and 285 down-regulated gens in STAT3cKO and WT mice hearts (Fold change≥2 and P < 0.01). (C) The top 20 of pathway enrichment were ananlyzed by Kyoto Encyclopedia of Genes and Genomes analysis. (D) The enrichment plot of cGMP-PKG signaling pathway, ECM-RECEPTOR interaction, and PI3K-AKT signaling pathway enrichment were ananlyzed by Gene Set Enrichment Analysis. (E) Western blot analysis showed the levels of PKG, P-PLB and P-TNI in WT mice hearts (n = 3) and STAT3cKO mice hearts (n = 3). Compared with WT group, *P < 0.05 by Student's unpaired t-test. **P < 0.01 by Student's unpaired t-test.