Research ArticlePhysiology

Cardiomyocyte glucocorticoid and mineralocorticoid receptors directly and antagonistically regulate heart disease in mice

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Science Signaling  16 Apr 2019:
Vol. 12, Issue 577, eaau9685
DOI: 10.1126/scisignal.aau9685
  • Fig. 1 Generation and survival of mice with conditional knockout of GR, MR, or both GR and MR in cardiomyocytes.

    (A) Mice deficient in cardiomyocyte GR (cardioGRKO), MR (cardioMRKO), or both GR and MR (cardioGRMRdKO) were generated by crossing mice with floxed GR and/or MR alleles with mice expressing Cre recombinase only in cardiomyocytes (αMHC-Cre). (B) Reverse transcription polymerase chain reaction (RT-PCR) of GR and MR mRNA from hearts of 2-month-old knockout mice and their littermate controls. Data are means ± SEM (n = 4 to 6 mice per group). Student’s t test was performed to determine significance. **P < 0.01 and ***P < 0.001 for GRKO compared to GRflox, for MRKO compared to MRflox, and for dKO compared to dflox. (C) Representative immunoblots of GR and MR protein from hearts of 3-month-old knockout mice and their littermate controls (n = 3 independent experiments). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) Survival curves for GRflox (n = 36), cardioGRKO (n = 92), MRflox (n = 26), cardioMRKO (n = 54), dflox (n = 41), and cardioGRMRdKO (n = 85) mice. Mantel-Cox log-rank test was performed with a Bonferroni-corrected threshold to determine significance. ***P < 0.001 for GRKO compared to GRflox. ###P < 0.001 for MRKO compared to GRKO and for dKO compared to GRKO.

  • Fig. 2 CardioGRMRdKO mice are protected from LV remodeling.

    (A) BW, HW, and HW/BW ratios were determined for 2-month-old control and knockout mice. Data are means ± SEM (n = 5 to 9 mice per group). (B) Cross-sectional area of LV cardiomyocytes in 2-month-old control and knockout hearts. Left panel shows representative confocal images of heart sections stained with fluorescein isothiocyanate (FITC)–lectin. Scale bars, 20 μm. Right panel shows quantitation of cardiomyocyte cross-sectional area. Data are means ± SEM (greater than 400 cardiomyocytes from n = 4 to 5 mice per group). (C and D) Representative images of intact hearts (left) and longitudinal H&E-stained heart sections (right) from 3-month-old (C) and 6-month-old (D) control and knockout mice. Scale bars, 2 mm. Images are representative of three to six mice per genotype. A one-way analysis of variance (ANOVA) was performed to determine significance. *P < 0.05, **P < 0.01, and ***P < 0.001 for GRKO compared to GRflox and for dKO compared to dflox. ###P < 0.001 for dKO compared to GRKO.

  • Fig. 3 CardioGRMRdKO mice are protected from LV systolic dysfunction.

    (A) Representative M-mode images from 3-month-old control and knockout mice. (B and C) Echocardiographic measurements of percent ejection fraction (EF) and percent fractional shortening (FS) were determined from transthoracic M-mode tracings made on control and knockout mice that were 3 months (B) or 6 months (C) old. Data are means ± SEM (n = 5 to 11 mice per group). A one-way ANOVA was performed to determine significance. **P < 0.01 and ***P < 0.001 for GRKO compared to GRflox and for Cre compared to wild type (WT). ###P < 0.001 for Cre compared to GRKO, for MRKO compared to GRKO, and for dKO compared to GRKO.

  • Fig. 4 Genes associated with cardiac pathology are dysregulated in cardioGRMRdKO hearts.

    Total RNA was isolated from whole hearts of 2-month-old control and knockout mice. (A) Myh7, Acta1, Nppb, and Acta2 mRNA levels were measured by RT-PCR. Data are means ± SEM (n = 8 to 11 mice per group). (B) Dmd, Ryr2, Klf15, and Ptgds mRNA levels were measured by RT-PCR. Data are means ± SEM (n = 8 to 12 mice per group). A one-way ANOVA was performed to determine significance. *P < 0.05, **P < 0.01, and ***P < 0.001 for GRKO compared to GRflox, for MRKO compared to MRflox, and for dKO compared to dflox. ##P < 0.01 and ###P < 0.001 for dKO compared to GRKO.

  • Fig. 5 Global gene expression profile in 1-month-old cardioGRKO, cardioMRKO, and cardioGRMRdKO hearts.

    Microarrays were performed on RNA isolated from the hearts of 1-month-old control and knockout mice. (A) Total number of genes differentially expressed in the hearts of knockout mice compared to their control littermates. (B) Differentially expressed genes in the knockout hearts were compared using a Venn diagram. (C) Diseases and disorders most significantly associated with the dysregulated genes in the knockout hearts as determined by IPA. (D) Gene enrichment comparison analysis of the dysregulated genes associated with “Cardiovascular Disease” in the three knockout hearts was performed using IPA. The disease annotations with a significant activation z score (absolute value ≥ 2) are shown. The retrieved annotation “Failure of Heart” was only significantly associated with the dysregulated genes in the cardioGRKO heart. ns, not significant.

  • Fig. 6 CardioGRMRdKO hearts are protected from alterations in Ca2+handling and oxidative stress observed in cardioGRKO hearts.

    (A) Signaling pathways most significantly associated with the dysregulated genes in 1-month-old knockout hearts as determined by IPA. (B) The “Cardiac β-Adrenergic Signaling” pathway overlaid with dysregulated genes in 1-month-old knockout hearts. Red and green colors correspond to up-regulation and down-regulation, respectively. (C and D) mRNA levels for the Ca2+ handling genes Cacna1c, Atp2a2, and Slc8a1 (C) and the oxidative stress genes Ncf1, Rac2, and Spp1 (D) were measured by RT-PCR in 3-month-old control and knockout hearts. Data are means ± SEM (n = 7 to 10 mice per group). *P < 0.05 and ***P < 0.001 for GRKO compared to GRflox, for MRKO compared to MRflox, and for dKO compared to dflox.

  • Fig. 7 CardioGRMRdKO hearts are protected from cell death observed in cardioGRKO hearts.

    (A) Molecular and cellular functions most significantly associated with the dysregulated genes in 1-month-old knockout hearts as determined by IPA. (B) Dysregulated genes associated with Cell Death and Survival in the knockout hearts were compared using a Venn diagram. (C) Gene enrichment comparison analysis of the dysregulated genes associated with Cell Death and Survival in the three knockout hearts was performed using IPA. The functional annotations with a significant activation z score (absolute value ≥ 2) are shown. The retrieved annotations were only significantly associated with the dysregulated genes in the cardioGRKO heart. (D) Analysis of cell death in knockout hearts. Left panel shows representative image of TUNEL-positive nuclei (arrow) in LV myocardium of 6-month cardioGRKO heart. Quadruple staining was performed: TUNEL (green), wheat germ agglutinin (WGA) (red), cardiac troponin T (Tnnt2) (cyan), and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 10 μm. Right panel shows quantitation of TUNEL-positive nuclei in 6-month knockout hearts. Data are means ± SEM (n = 4 to 6 mice per group). A one-way ANOVA was performed to determine significance. *P < 0.05 for GRKO compared to GRflox.

  • Fig. 8 Gene changes associated with cardioprotection are uniquely observed in cardioGRMRdKO hearts.

    Total RNA was isolated from whole hearts of 1-month-old (A) and 2-month-old (B) control and knockout hearts. Agt, Ccnd2, Hdac4, and Ankrd23 mRNA levels were measured by RT-PCR. Data are means ± SEM (n = 4 to 9 mice per group). A one-way ANOVA was performed to determine significance. *P < 0.05, **P < 0.01, and ***P < 0.001 for GRKO compared to GRflox, for MRKO compared to MRflox, and for dKO compared to dflox. #P < 0.05, ##P < 0.01, and ###P < 0.001 for dKO compared to GRKO. &P < 0.05, &&P < 0.01, and &&&P < 0.001 for dKO compared to MRKO.

  • Fig. 9 Reexpression of MR in the cardioGRMRdKO heart reverses cardioprotective gene changes.

    CardioGRMRdKO mice and their littermate controls (dflox) were injected intravenously with PBS, AAV-Tnnt2-GFP, or AAV-Tnnt2-MR at 4 to 6 weeks of age. (A) Representative immunoblot shows MR and GFP expression in hearts isolated from injected mice that were 6 months old (n = 3 independent experiments). Positive (Pos) and negative (Neg) controls are hippocampal lysates from a wild-type mouse and a littermate mouse with conditional knockout of MR in the hippocampus, respectively. (B) RT-PCR analysis of Agt, Ccnd2, Hdac4, and Ankrd23 mRNA levels in hearts from injected dflox and cardioGRMRdKO mice that were 6 months old. Data are means ± SEM (n = 7 to 10 mice per group). (C) RT-PCR analysis of Myh7 and Nppb mRNA levels in hearts from injected dflox and cardioGRMRdKO mice that were 6 months old. Data are means ± SEM (n = 7 to 10 mice per group). A one-way ANOVA was performed to determine significance. aP < 0.05, compared to dflox + PBS. bP < 0.05, compared to dflox + GFP. cP < 0.05, compared to dflox + MR. dP < 0.05, compared to dKO + PBS. eP < 0.05, compared to dKO + GFP.

  • Fig. 10 Cardiomyocyte GR and MR signaling and heart disease.

    Findings from our genetic mouse models suggest that both insufficient cardiomyocyte GR signaling and deleterious cardiomyocyte MR signaling contribute to heart disease. A deficiency in cardiomyocyte GR signaling alone (−GR) leads to myocardial stress (lightning bolt) and mild hypertrophy in both the cardioGRKO and cardioGRMRdKO hearts. In contrast, a deficiency in cardiomyocyte MR signaling alone (−MR) does not have an overt effect. In the cardioGRKO hearts, cardiomyocyte MR signaling becomes deleterious and exacerbates the hypertrophic response, leading to maladaptive remodeling and heart failure. In the cardioGRMRdKO hearts, the mild cardiac hypertrophy triggered by the loss of cardiomyocyte GR signaling is not exacerbated in the absence of cardiomyocyte MR signaling and heart function is preserved for a longer period of time.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/577/eaau9685/DC1

    Fig. S1. Knockout of GR and/or MR is specific to the heart.

    Fig. S2. Systolic blood pressure in cardioGRKO, cardioMRKO, and cardioGRMRdKO mice.

    Fig. S3. Histological analysis of cardioGRKO and cardioGRMRdKO hearts.

    Fig. S4. CardioGRMRdKO mice exhibit RV hypertrophy but no lung edema at 6 months of age.

    Fig. S5. Genes associated with cardiac pathology are dysregulated in cardioGRMRdKO hearts.

    Fig. S6. Global gene expression profile in 2-month-old cardioGRKO, cardioMRKO, and cardioGRMRdKO hearts.

    Fig. S7. Heat maps of differentially expressed genes in the cardioGRKO, cardioMRKO, and cardioGRMRdKO hearts.

    Fig. S8. Dysregulation of Cell Death and Survival genes in the cardioGRKO, cardioMRKO, and cardioGRMRdKO hearts.

    Fig. S9. CardioGRMRdKO hearts are protected from the cell death observed in cardioGRKO hearts.

    Fig. S10. AAV constructs using the Tnnt2 promoter are expressed specifically in cardiomyocytes.

    Table S1. Electrocardiographic analysis of cardioGRKO, cardioMRKO, and cardioGRMRdKO mice.

    Table S2. Echocardiography on 3-month-old αMHC-Cre, cardioGRKO, cardioMRKO, and cardioGRMRdKO mice.

    Table S3. Echocardiography on 1-month-old cardioGRKO mice.

    Table S4. Echocardiography on 6-month-old αMHC-Cre, cardioGRKO, cardioMRKO, and cardioGRMRdKO mice.

    Table S5. Echocardiography on cardioGRMRdKO mice injected with AAV-Tnnt2-MR.

    Data file S1. Differentially expressed genes in 1-month cardioGRKO, cardioMRKO, and cardioGRMRdKO hearts.

    Data file S2. Differentially expressed genes in 2-month cardioGRKO, cardioMRKO, and cardioGRMRdKO hearts.

  • The PDF file includes:

    • Fig. S1. Knockout of GR and/or MR is specific to the heart.
    • Fig. S2. Systolic blood pressure in cardioGRKO, cardioMRKO, and cardioGRMRdKO mice.
    • Fig. S3. Histological analysis of cardioGRKO and cardioGRMRdKO hearts.
    • Fig. S4. CardioGRMRdKO mice exhibit RV hypertrophy but no lung edema at 6 months of age.
    • Fig. S5. Genes associated with cardiac pathology are dysregulated in cardioGRMRdKO hearts.
    • Fig. S6. Global gene expression profile in 2-month-old cardioGRKO, cardioMRKO, and cardioGRMRdKO hearts.
    • Fig. S7. Heat maps of differentially expressed genes in the cardioGRKO, cardioMRKO, and cardioGRMRdKO hearts.
    • Fig. S8. Dysregulation of Cell Death and Survival genes in the cardioGRKO, cardioMRKO, and cardioGRMRdKO hearts.
    • Fig. S9. CardioGRMRdKO hearts are protected from the cell death observed in cardioGRKO hearts.
    • Fig. S10. AAV constructs using the Tnnt2 promoter are expressed specifically in cardiomyocytes.
    • Table S1. Electrocardiographic analysis of cardioGRKO, cardioMRKO, and cardioGRMRdKO mice.
    • Table S2. Echocardiography on 3-month-old αMHC-Cre, cardioGRKO, cardioMRKO, and cardioGRMRdKO mice.
    • Table S3. Echocardiography on 1-month-old cardioGRKO mice.
    • Table S4. Echocardiography on 6-month-old αMHC-Cre, cardioGRKO, cardioMRKO, and cardioGRMRdKO mice.
    • Table S5. Echocardiography on cardioGRMRdKO mice injected with AAV-Tnnt2-MR.
    • Legends for data files S1 and S2

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Differentially expressed genes in 1-month cardioGRKO, cardioMRKO, and cardioGRMRdKO hearts.
    • Data file S2 (Microsoft Excel format). Differentially expressed genes in 2-month cardioGRKO, cardioMRKO, and cardioGRMRdKO hearts.

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