Glucocorticoid chronopharmacology promotes glucose metabolism in heart through a cardiomyocyte-autonomous transactivation program

Figures

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  Figure 1. Light-phase glucocorticoid stimulation improves glucose uptake and oxidation in the heart. (A) Gene Ontology (GO) analysis revealed that ZT0 intermittent prednisone increased expression of Klf15, Adipor1, Mpc1, and Mpc2 from the enriched pathways of pyruvate transport and glucose import in the WT hearts. (B) Upregulation of KLF15 by prednisone was higher after ZT0 than ZT12 dosing, and upregulation of Adipor1, Mpc1, and Mpc2 was specific to ZT0 and blunted by ZT12 dosing. (C) Co-IPs in heart tissue showed that KLF15 interaction with GR was higher with ZT0 than ZT12 prednisone. (D) Consistent with Adipor1 upregulation, ZT0 prednisone treatment decreased myocardial ceramide levels and increased insulin-dependent 2DG uptake in the heart. (E) After ZT0, but not ZT12, treatment, cardiomyocytes increased basal glucose-fueled respiration ex vivo (arrow) and ATP production. (F) Consistent with Mpc1/2 upregulation, ZT0 treatment increased ADP-stimulated respiration (arrow) and respiratory control ratio with pyruvate in isolated mitochondria. (G) Circadian time-course qPCR analyses in myocardial tissues revealed oscillations of variable amplitude for Klf15, Adipor1, and Mpc1/2 expression.…

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  Figure 2. ChIP-seq and RNA-seq datasets reveal nonredundant mediator effect of both cardiomyocyte-specific GR and KLF15 on ZT0 glucocorticoid gene programs in the heart. (A) Validation of ChIP-seq datasets through unbiased motif analysis. (B) Prednisone increased peak numbers and canonical epigenomic peak signal (arrows) for both GR and KLF15 without changes in relative enrichment in genomic localization, which was enriched in promoter and 5′ UTRs for both factors. (C) PCA analyses show sample clustering according to drug and genotype variables. n = 3 ♂/group. In B and C, Welch’s t test was used to assess peak number and 2-way ANOVA with Šidák’s post hoc test was used to evaluate peak localization. *P < 0.05.

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  Figure 3. Cardiomyocyte GR and KLF15 are required for the ZT0 prednisone effects on gene transactivation, insulin sensitization, and pyruvate oxidation in heart. (A) GR and KLF15 showed steroid-sensitive peaks on the Adipor1 TSS (arrows). Drug-induced upregulation was blunted by inducible cardiomyocyte-specific ablation of either GR or KLF15. (B) Transcriptional/metabolic effects of treatment appeared durable beyond the resting phase, as GR and KLF15 were both required for the treatment effects on myocardial ceramide content reduction and insulin-stimulated glucose uptake increment in the heart in the active phase (ZT16). (C and D) Similarly, GR and KLF15 were required for the drug effect on Mpc1/2 transactivation, and for the chronic treatment effects on pyruvate oxidation in the heart in the active phase. Data are presented as mean ± SEM; histograms also show individual mouse values. n = 3 ♂/group in A and C; n = 8 ♂/group in B and D. *P < 0.05; **P < 0.01; ****P < 0.0001 by 2-way ANOVA with Šidák’s post hoc test.

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  Figure 4. Functional cardiac assessments for chronic ZT0 prednisone effects on uninjured hearts after cardiomyocyte gene ablation. Either GR-KO or KLF15-KO induced significant losses in fractional shortening, stroke volume, and gains in heart weight/tibia length compared with WT. In WT uninjured hearts, treatment increased stroke volume and fractional shortening, with no significant trends in heart weight. As expected, treatment had no sizable effects in KO hearts. Data are presented as mean ± SEM; histograms also show individual mouse values. n = 6 ♂/group. *P < 0.05; **P < 0.01; ****P < 0.0001 by 3-way ANOVA with Šidák’s post hoc test (curves) or 2-way ANOVA with Šidák’s post hoc test (histograms).

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  Figure 5. ZT0 glucocorticoid treatment rescues glucose oxidation and diastolic function in db/db mice. (A) Schematic showing the 12-week-long prednisone treatment of db/db and their littermate controls (db/+) at ZT0 and ZT12. (B) Representative images of pulse wave Doppler echocardiography for mitral valve flow, and quantitation of baseline defects in E/e′, stroke volume, and heart mass in db/db mice. (C) ZT0 treatment decreased E/e′ in db/db hearts to db/+-like levels, while ZT12 treatment had no sizable effects. (D) ZT0, but not ZT12, treatment increased stroke volume in both db/db and db/+ mice. (E) Cardiac hypertrophy was alleviated in db/db hearts by ZT0, but not ZT12 treatment. (F) Treatment increased glucose uptake in myocardial tissue, measured as 2DG uptake, in both db/+ and db/db hearts only with ZT0 dosing. (G) Treatment increased glucose-fueled ATP production calculated from Seahorse curves in isolated cardiomyocytes in both db/+ and db/db mice according to time of intake (ZT0 but not ZT12), increasing db/db values to control-like levels. Data are presented as mean ± SEM; histograms also show individual mouse values. n = (5 ♀ + 5 ♂)/group. ****P < 0.0001 by Welch’s t test (B)…

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  Figure 6. Knockdown of GR and KLF15 in db/db hearts in vivo blocks the ZT0 prednisone effects. (A) Representative Western blot showing knockdown of GR and KLF15 at 12 weeks after transduction and ZT0 prednisone regimen start, as well as blunting of the treatment effect on ADIPOR1, MPC1, and MPC2 protein level upregulation in hearts of db/db mice transduced with the knockdown vectors compared with mice transduced with scramble vectors. (B and C) Knockdown MyoAAV combination blocked the treatment effects on diastolic dysfunction (E/e′), stroke volume, cardiac hypertrophy (heart weight/tibia length), and insulin-driven glucose uptake (2DG6P). (D) Treatment increased glucose-fueled respiration (oxygen consumption rate, OCR) in permeabilized cardiac biopsies and pyruvate-fueled respiration (respiratory control ratio, RCR) in isolated myocardial mitochondria in scramble-, but not knockdown-transduced, hearts. Data are presented as mean ± SEM; histograms also show individual mouse values. n = 5 ♂/group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 2-way ANOVA with Šidák’s post hoc test.