Fig 2: BMAL1 is required for the effects of intermittent light-phase prednisone on muscle bioenergetics.Results are shown after a 12-week-long treatment with intermittent once-weekly prednisone with dosing restricted to ZT0 (light phase) versus ZT14 (dark phase). (A) In WT mice, compared to isochronic vehicle controls, ZT0, but not ZT14, prednisone improved treadmill performance, muscle fatigue (in situ tibialis anterior), and body-wide VO2 normalized to lean mass. (B and C) This correlated with gains in muscle NAD+ (mass spectrometry) and basal OCR in muscle tissue (Seahorse respirometry) after ZT0, but not ZT14, regimens. (D) ZT0, but not ZT14, prednisone increased in muscle catabolism of either glucose or palmitate, as shown by steady-state 13C tracing in ex vivo contracting muscle. (E to G) Compared to BMAL1-WT littermates, BMAL1-KO mice blunted the gain in muscle NAD+ induced by ZT0 prednisone. This correlated with analogous trends in basal respiration and 13C-labeled nutrient oxidative catabolism in muscle tissue. (H) The effects of ZT0 prednisone treatment on treadmill performance, muscle fatigue, and VO2 in BMAL1-WT mice were blocked in BMAL1-KO mice. n = 5 (male, female) per group for (A) to (D); n = 3 (male, female) per group for (E) to (I). *P < 0.05, one-way analysis of variance (ANOVA) + Sidak (histograms) and two-way ANOVA (curves). ns, not significant.

Fig 3: Light-phase intermittent prednisone activates a circadian-metabolic transcriptional program in the infarcted heart. WT mice were randomized into sham or myocardial infarction (MI) cohorts and treated with light-phase intermittent 1 mg/kg prednisone for 6 weeks from 2-weeks post–MI. (A) Treatment improved fractional shortening and stroke volume in mice with MI. (B) PCA analysis of RNA-seq profiles showed treatment-associated clustering in both sham and MI hearts. (C) Differentially expressed (DE) genes in treated versus control hearts showed 89% overlap in GO analysis between sham and MI conditions. Among overlapping GO terms were inflammation and circadian rhythm pathways. (D) Chart presenting genes with concordant versus discordant regulation by treatment in sham versus MI conditions. Concordant genes are on the right side, while discordant genes are on the left side. Activating clock complex genes (Arntl, Clock, Rora), as well as metabolic regulators Nampt, Ppargc1a and Ppara were among concordant genes. Repressive clock complex genes (Nr1d1, Per1-3) were among discordant genes. (E–F) Treatment increased mitochondrial respiration (tissue OCR and mitochondrial RCR) and mitochondrial abundance (Mitotracker fluorometry in myocardial tissue and mtDNA quantitation) in both sham and MI hearts. * = p < 0.05, ** = p < 0.01, *** = p < 0.001; 2w ANOVA (curves in A), 1w ANOVA + Sidak (A, E-F); Benjamini-Hochberg for GO term FDR (C).

Fig 4: The mitochondrial effects of light-phase prednisone are dependent upon cardiomyocyte-specific GR and BMAL1 expression. (A) At 24-hours post-injection, light-phase prednisone increased mitochondrial respiration in GR-WT but not GR-KO hearts (induced cardiomyocyte-specific GR ablation in postnatal tissue), as compared to respective vehicle controls. (B–C) Analogous trends were found in myocardial content of NAD+ and ATP (mass-spec), as well as expression levels of Nampt and Ppargc1a. (D–F) Analogous trends in myocardial basal respiration, RCR in isolated mitochondria, NAD+ and ATP content (mass-spec), and Nampt and Ppargc1a expression were found comparing BMAL1-WT to BMAL1-KO hearts (induced cardiomyocyte-specific BMAL1 ablation in postnatal tissue). * = p < 0.05, ** = p < 0.01, *** = p < 0.001; 1w ANOVA + Sidak.

Fig 5: Light-phase but not dark-phase injection of pulsatile or intermittent prednisone increases biomarkers of mitochondrial metabolism in the myocardium. (A–B) Mice were injected with one dose of 1 mg/kg i.p. prednisone in vivo and sampled at 1-, 5-, 9-, 13-, 17- and 21-hours post-pulse. Light-phase prednisone (pulse at ZT0) increased NAD+ and ATP levels as assessed through mass-spec (A), as well as Nampt and Ppargc1a expression (B), in hearts in vivo in two circadian phases following injection (arrows). These effects were blocked with a dark-phase drug pulse (ZT12). (C) The light-phase prednisone pulse correlated with increased amplitude of Arntl (BMAL1) and Clock expression (activating clock complex), while the dark-phase prednisone transiently elevated Per1 and Cry2 gene expression (repressive clock complex) during their circadian trough. (D–H) Mice were injected with once-weekly 1 mg/kg prednisone for 12 weeks at either ZT0 (light-phase treatment) or ZT12 (dark-phase). Compared to isochronic vehicle treatments, light-phase but not dark-phase treatments improved NAD+ and ATP levels in myocardium, as quantitated through mass-spec (D). This correlated with increased mitochondrial abundance, estimated by mtDNA/nDNA ratio and Mitotracker fluorometry in tissue, and decreased ROS levels, estimated through MitoSOX fluorometry (E). TEM imaging showed increased mitochondria number and mitochondria-filled areas in light-phase- but not dark-phase-treated myocardial sections (F). Seahorse respirometry in isolated cardiomyocytes showed increased glucose- or lipid-fueled basal respiration and ATP production after light-phase but not dark-phase treatment (G). These trends correlated with RCR trends in isolated mitochondria with either pyruvate or palmitoylcarnitine (H). * = p < 0.05, ** = p < 0.01, *** = p < 0.001; 2w ANOVA (curves in A-B, G), 1w ANOVA + Sidak (D–H).