BH4 activates CaMKK2 and rescues the cardiomyopathic phenotype in rodent models of diabetes

Figures

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  Figure 1.. BH4 supplementation recovers cardiac systolic dysfunction in a rat model of late-stage type 2 diabetes mellitus. (A) Experimental design. (B, C) Body weight (B) and i.p. glucose-tolerance tests (n = 5/group) (C). (D) Representative M-mode echocardiography at 50 wk. (E) Fractional shortening during pretreatment and after 1 and 2 wk of BH4 or vehicle treatment (n = 5/group). (F) 2D echocardiography results. (G) Comparison of radial strain (n = 5/group). (H) Average traces of electrical stimulation–induced sarcomere shortening in isolated left ventricle (LV) cardiomyocytes (n = 15 cells from three animals/group). (I) Peak shortening of isolated LV cardiomyocytes (n = 15/group). (J) Velocity of shortening and Re-lengthening of isolated LV cardiomyocytes (n = 15/group). *P < 0.05 versus Long–Evans Tokushima Otsuka; #P < 0.05 versus Otsuka Long–Evans Tokushima Fatty.

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  Figure S1.. Lipid profiling in blood serum of Long–Evans Tokushima Otsuka, Otsuka Long–Evans Tokushima Fatty (OLETF), and OLETF/BH4 rats. LDL, low-density lipoprotein.

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  Figure S2.. BH4 supplementation enhances cardiac contractility in db/db mice. (A) M-mode echocardiography of WT, db/db, and db/db-BH4 groups. (B) Comparison of the ejection fraction before and after BH4 supplementation or vehicle treatment. (C) Comparison of fractional shortening before and after BH4 supplementation or vehicle treatment (each dot indicates a tested animal). *P < 0.05, db/db versus db/db-BH4.

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  Figure 2.. BH4 supplementation recovers pathological cardiac remodeling in a model of late-stage type 2 diabetes mellitus. (A) Representative H&E staining of cross-sectioned hearts. (B) Cross-sectional size of cardiac muscle fibers. (C) Masson’s trichrome staining of cross-sectioned hearts. (D) Quantification of fibrotic area. (E) Images of TUNEL-stained heart tissues from experimental rats. Red arrow: TUNEL+ apoptotic cells. Scale bar: 50 μm. (F) Quantification of apoptotic cell death. Data in (A, B, C, D, E, F) represent the mean ± SEM (n = 4 animals/group). *P < 0.05 versus Long–Evans Tokushima Otsuka; #P < 0.05 versus Otsuka Long–Evans Tokushima Fatty. TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

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  Figure S3.. BH4 suppresses proteasomal activity and fibrosis in Otsuka Long–Evans Tokushima Fatty rats. (A) Zymographic analysis comparing matrix metalloproteinase (MMP) activity in the hearts of experimental animals. (B) Relative levels of MMP-2, ProMMP-2, and ProMMP-9. (C) 26S proteasome activity in cardiac tissues from model animals. Lactacystin, a 26S proteasome inhibitor, was used as a positive control. (D) Immunoblot images of selected pro-fibrotic proteins. (E) Relative protein levels of inactive TGF-β1, active TGF-β1, fibronectin, collagen I, collagen III, and monocyte chemoattractant protein-1 normalized against β-tubulin level. TGF-β1, monocyte chemoattractant protein-1, and collagen I and III bands were cropped from the same gel, and fibronectin and β-actin bands were cropped from another gel. Both gels ran simultaneously with 20 μg of each protein (n = 3/group). (F) Immunoblot images and relative protein level of total and phopho-eNOS proteins. (n = 3/group) Data represent the mean ± SEM. *P < 0.05 versus Long–Evans Tokushima Otsuka; #P < 0.05 versus Otsuka Long–Evans Tokushima Fatty.

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  Figure 3.. BH4 supplementation improves the biochemical phenotype of diabetic rat hearts. (A) Total biopterin and BH4 level in heart and mitochondria (n = 4/group). (B) BH4/total biopterin ratios (n = 4/group). (C) Serum levels of CPK and LDH. (D) ATP levels. (E) Mitochondrial membrane potential. (F) Reactive oxygen species production assessed by relative Amplex Red intensity. (G) Lipid oxidation assay to measure oxidative stress in heart tissue. (H, I, J, K, L) Activities of OXPHOS complexes I–V. (M) Citrate synthase activity. *P < 0.05 versus Long–Evans Tokushima Otsuka; #P < 0.05 versus Otsuka Long–Evans Tokushima Fatty.

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  Figure S4.. BH4 improves mitochondrial ultrastructure in diabetic rat and mouse hearts. (A) Representative EM image of cardiac left ventricle tissue from Long–Evans Tokushima Otsuka (LETO), Otsuka Long–Evans Tokushima Fatty (OLETF), and OLETF/BH4 rats. (B) EM images of cardiac left ventricle tissue from WT, db/db, and db/db-BH4 mice. (C, D) Quantification of damaged mitochondrial proportion in hearts from LETO, OLETF, and OLETF/BH4 rats (C) and WT, db/db, and db/db-BH4 mice (n = 5/group). (C) *P < 0.05 versus LETO; #P < 0.05 versus OLETF. (D) *P < 0.05 versus WT; #P < 0.05 versus db/db.

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  Figure 4.. PGC-1α mediates the OXPHOS pathway and a primary BH4 target. (A) Differently expressed proteins between Otsuka Long–Evans Tokushima Fatty (OLETF) and OLETF/BH4 hearts and related cardio-relevant biological pathways. Red circles represent proteins directly linked to four biological pathways (direct links). Purple circles represent proteins that interact with red-circle proteins (second link). Blue circles represent proteins that interact with purple-circle proteins (third link). (B) Four biological pathways and their component proteins. (C) Enrichment P-values of four BH4 targets related to cardio-relevant biological pathways associated with heart function in the DCM model. (D, E) Immunoblot analysis of proteins related to mitochondrial biogenesis and functional regulation of (D) mitochondrial OXPHOS complexes (E) (n = 3/group). ATP5A for complex V, UQCRC2 for complex III, SDHB for complex II, and NDUFSB8 for complex I. Relative protein expression was normalized to β-actin. *P < 0.05 versus Long–Evans Tokushima Otsuka; #P < 0.05 versus OLETF. P/T, phosphorylated:unphosphorylated (total) protein ratio. Source data are available for this figure.

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  Figure S5.. BH4 treatment affects six major biological pathways. (A) Differently expressed proteins between Otsuka Long–Evans Tokushima Fatty (OLETF) and OLETF/BH4 hearts and related biological pathways. Red triangles represent biological pathways that were affected by BH4 treatment. Blue circles represent proteins that were differentially expressed between OLETF and OLETF/BH4 hearts. (B) Enrichment P-values of six BH4 targets related to biological pathways in the DCM model.

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  Figure S6.. In vitro kinase assay for screening BH4 target kinases.

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  Figure 5.. CaMKK2 is a novel BH4-binding target. (A, B, C, D, E, F) Protein–ligand docking simulation results for BH4–CaMKIV (A, B) and BH4–CaMKK2 (D, E). (C, F) Sensorgrams for BH4 binding to recombinant CaMKIV (C) or CaMKK2 (F).

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  Figure 6.. BH4 regulates PGC-1α levels and AMPK phosphorylation by modulating CaMKIV/CREB signaling. (A, B) Representative Western blot (A) and quantitation (B) of total and phosphorylated signaling proteins in animal hearts. β-tubulin served as an internal standard. *P < 0.05 versus WT; #P < 0.05 versus Spr−/− (n = 3/group). Source data are available for this figure.