Cardiomyocyte Janus kinase 1 (JAK1) signaling is required for cardiac homeostasis and cytokine-dependent activation of STAT3

Figures

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  Fig. 1.. Cardiomyocyte-specific deletion of Jak1. (A) Schematic of breeding scheme to generate mice lacking JAK1 in cardiomyocytes by crossing mice harboring a conditional null allele for Jak1 to α-MHC-Cre (Myh6-Cre) hemizygous transgenic mice to produce Jak1fl/fl, Myh6-Cre (Jak1cKO) mice with Jak1 deleted in cardiomyocytes. (B) Immunoblotting and quantification of relative (C) JAK1 and (D) JAK2 protein levels in whole cardiac lysates generated from the indicated genotypes of male and female mice at two months of age. n = 4 Jak1fl/fl and 5 Jak1cKO. JAK protein levels were normalized to Gapdh as a loading control. Assessment of (E) Jak1 mRNA levels by qPCR and (F) JAK1 protein levels by immunoblotting in adult cardiomyocytes (ACMs) isolated from hearts of 2 months old male and female mice. n = 4 in E and n = 3 in F. α-sarcomeric (sarc.) actin was used as a loading control for normalization of relative JAK1 protein levels shown on the immunoblotting panel in F. Data are presented as the mean value ± the standard error of the mean. *P < 0.05, ns, not significant, unpaired t-test.

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  Fig. 2.. Defective STAT3 signaling in Jak1-deleted cardiomyocytes. (A) Experimental assessment of JAK-STAT signaling in Jak1-deleted cardiomyocytes. Adult ventricular cardiomyocytes (ACMs) were isolated from male and female control (Jak1fl/fl) or Jak1-deleted (Jak1cKO) hearts at 2 months of age and treated with vehicle or oncostatin-M (OSM, 10 ng/mL) for 30 min before harvesting for Western blotting or fixing for immunocytochemistry to assess STAT3 phosphorylation and nuclear translocation, respectively, or for 16 h to assess gene expression. (B) Western blotting and quantification of (C) STAT3 phosphorylation at Tyr-705 normalized to total STAT3 and (D) total STAT3 protein normalized to Gapdh in control Jak1fl/fl or Jak1cKO ACMs with or without OSM treatment. n = 3. (E) Stat3 mRNA levels were quantified by qPCR in ACMs isolated from Jak1fl/fl or Jak1cKO hearts. n = 4. (F) Representative images of immunostaining for endogenous STAT3 (red) in control (Jak1fl/fl) or Jak1-deleted (Jak1cKO) ACMs with or without OSM treatment. Nuclei were stained blue with DAPI. Scale bar = 20 μm. (G) Relative Manders coefficients for colocalization of STAT3 with nuclear DAPI signal in ACMs of the indicated…

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  Fig. 3.. JAK1 is required for cytokine-stimulated transcriptional reprogramming in cardiac myocytes. (A) Experimental assessment of oncostatin-M (OSM)-inducible target genes. Adult ventricular cardiomyocytes (ACMs) isolated from male and female mice were treated with vehicle or OSM (10 ng/mL) for 16 h in culture, and RNA isolated for (B—C) RNA sequencing (RNAseq) or (D—H) qPCR. (B) Histogram of the top 10 KEGG biological processes most significantly enriched with upregulated genes in OSM-treated ACMs compared to vehicle-treated ACMs. Values at the end of histograms indicate the number of upregulated genes with OSM treatment in the pathway. Histograms for biological processes related to inflammation are colored light blue and the JAK-STAT pathway itself colored black. (C) Heat map depicting the 10 most-upregulated transcripts in OSM-treated ACMs relative to vehicle-treated ACMs by RNAseq. Relative mRNA levels of (D) Thbd, (E) Timp1 (F) Socs3, (G) Sprr1a, (H) Serpinb1a, and (I) Vegfa were assessed by qPCR in ACMs isolated from male and female mice of the indicated genotypes and treated with vehicle or OSM. n = 3 Myh6-Cre, 3 Jak1fl/fl, and 4 Jak1cKO in D, E, H, and I; n = 3 Myh6-Cre, 4…

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  Fig. 4.. Cardiomyocyte-specific loss of JAK1 results in dilated cardiomyopathy. (A-K) Assessment of cardiac structure and function in conditional-null Jak1 mice with cardiomyocyte-specific loss of JAK1 (Jak1fl/fl, Myh6-Cre, Jak1cKO) compared to control genotypes expressing the conditional-null allele (Jak1fl/fl) or Jak1+/+, Myh6-Cre mice expressing the Cre transgene alone (Myh6-Cre) at (A-D) 2 months and (E-H) 6 months of age. Quantification of (A, E) diastolic left ventricular (LV) posterior wall thickness (LVPWd), (B, F) LV internal diameter (LVID) during diastole (LVIDd), (C, G) systolic LVID (LVIDs), and percent (D, H) ejection fraction (E.F.) in the indicated genotypes of mice of both male and female mice. (I) Representative M-mode images of the indicated genotypes of male mice at 6 months of age. n = 6 Myh6-Cre, 5 Jak1fl/fl, and 6 Jak1cKO in A-D and n = 8 Myh6-Cre, 13 Jak1fl/fl, and 18 Jak1cKO in E-H. Data are presented as the mean value ± the standard error of the mean. *P < 0.05, **P < 0.01, one-way ANOVA with post-hoc Tukey’s multiple comparisons test. Also see Supplemental Table S1 and Table S2.

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  Fig. 5.. Pathological cardiac hypertrophy and myocardial fibrosis in aged mice with cardiomyocyte-specific loss of JAK1. (A) H&E histological staining of cardiac sections (scale bar = 1 mm) of male mice and (B) heart weight-to-body weight ratios (HW/BW) of the indicated genotypes of male and female mice at 8 months of age. n = 13 Myh6-Cre, 13 Jak1fl/fl, and 17 Jak1cKO. (C) Representative images of Picro Sirius Red-stained cardiac sections (scale bar = 100 μm) and (D) quantification of interstitial myocardial fibrosis in male and female mice of the indicated genotypes at 8 months of age. n = 12 Myh6-Cre, 13 Jak1fl/fl, and 16 Jak1cKO. Representative images in C are mixed sexes (Myh6-Cre male, Jak1fl/fl and Jak1cKO female). Cardiac transcript levels of (E) Postn and (F) Nox4 quantified by qPCR in male and female mice. n = 11 Myh6-Cre, 13 Jak1fl/fl, and 17 Jak1cKO. (G) Representative images of immunohistochemistry for the fibroblast marker platelet-derived growth factor receptor-α (PDGFRα) (red) in cardiac sections of 8 months old male mice. Cardiomyocytes were stained with phalloidin (F-actin, green) and nuclei stained with DAPI (blue). Scale bar = 100 μm. (H, I) Western blotting and (J, K)…