Fructose is a major component of dietary sugar and its overconsumption exacerbates key pathological features of metabolic syndrome. The central fructose-metabolising enzyme is ketohexokinase (KHK), which exists in two isoforms: KHK-A and KHK-C, generated through mutually exclusive alternative splicing of KHK pre-mRNAs. KHK-C displays superior affinity for fructose compared with KHK-A and is produced primarily in the liver, thus restricting fructose metabolism almost exclusively to this organ. Here we show that myocardial hypoxia actuates fructose metabolism in human and mouse models of pathological cardiac hypertrophy through hypoxia-inducible factor 1α (HIF1α) activation of SF3B1 and SF3B1-mediated splice switching of KHK-A to KHK-C. Heart-specific depletion of SF3B1 or genetic ablation of Khk, but not Khk-A alone, in mice, suppresses pathological stress-induced fructose metabolism, growth and contractile dysfunction, thus defining signalling components and molecular underpinnings of a fructose metabolism regulatory system crucial for pathological growth.
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We thank S. Georgiev, T. Simka, S. Xu, C. Bischoff, J. M. Dominguez, W. Kovacs and M. Piontek and other members of the Krek laboratory for discussions, help and technical assistance. We are grateful to M. Stoffel for performing tail vein injections. K. Chien, A. Asipu and R. J. Johnson provided mouse lines. This work was supported by grants from Sinergia (Swiss National Science Foundation) to W.K., T.P. and J. U. and the Swiss Heart Foundation to W.K.
The authors declare no competing financial interests.
Extended data figures and tables
a, Schematic experimental workflow. b, Comparison of C1QBP, HNRNPH3, JMJD6 and SF3B1 promoter sequences of human and mouse illustrate putative HREs up to 1,000 bp upstream of the transcription start site (TSS). The core consensus HRE motif is shown in colour. c, d, qPCR (c) and immunoblotting (d) for Sf3b1 expression in NMCs transduced with scrambled shNs viruses or viruses bearing two distinct shRNAs against Sf3b1. All values obtained by qPCR are presented in relation to Sf3b1 expression in shNs (set as 1.0) (n = 3 biological replicates per group). e, RT–PCR analysis of Khk mRNA as in Fig. 1g in NMCs transduced as indicated. f, RT–PCR analyses of Khk mRNA from NMCs infected as indicated and stimulated with mock or isoproterenol (ISO). PCR products were digested with HincII as in Fig. 1c . Splicing is quantified as percent of the Khk-C isoform (%Khk-C). g, RT–PCR based assessment of Khk-A and Khk-C isoform expression as done in Fig. 1g. h, i, Ventricular samples of sham- or 1K1C-operated mice were analysed for Khk isoform expression (h) as done in Fig. 1g or Sf3b1, Khk-A and Khk-C mRNA expression (i) by qPCR; n = 6 for each group. j, k, Ventricular samples of sham- or TAC-treated mice (j) and NaCl- or isoproterenol-treated mice (k) were analysed for expression of Sf3b1, Khk-A and Khk-C mRNA expression by qPCR (n = 6 for each individual group). l, NMCs transduced as specified were validated for protein expression of indicated proteins by using immunoblots. In addition lysates of NMCs overexpressing KHK-A or KHK-C were processed for immunoblotting Khk antibodies. m, n, Ventricular samples of sham- or TAC-treated (m) and sham- or isoproterenol-treated mice (n) were analysed for indicated proteins by immunoblotting. o, Probes from left ventricles of 1K1C-, TAC- or isoproterenol-treated mice and their corresponding controls were analysed for mRNA expression of Hif1α targets. All values were presented in relation to sham-operated controls set as 1.0 (n = 6 for each individual group). p, q, Left-ventricular biopsies of HCM patients and healthy controls were analysed for mRNA expression of SF3B1, KHK-A and KHK-C (p) and HIF1α targets (q). All values are presented in relation to healthy controls set as 1.0 (n = 6 for controls and n = 16 for patient samples). Error bars are s.d. (c) or s.e.m. (i–k, o–q). *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed unpaired t-test (c, i–k, o–q).
Extended Data Figure 2 SF3B1 is a HIF1α-target gene, whose product regulates growth and metabolic shift in vitro.
a, NMCs infected as denoted, were stimulated with either mock, phenylephrine and isoproterenol, respectively. Sf3b1 mRNA expression was examined by qPCR. (n = 3 biological replicates). b, NMCs were transduced as indicated and processed for qPCR analysis of Sf3b1 mRNA. (n = 3 biological replicates). c, Comparative analysis of a conserved HRE (shown in red) in the promoter of SF3B1 of diverse mammalian species located 122 bp downstream of the transcription start site (TSS). d–g, Immunoblot detection of indicated proteins of NMCs transduced with shNs, and shSf3b lentiviruses treated with phenylephrine or isoproterenol (d) co-infected with empty vector or HIF1αΔODD (e) cultured under normoxic (21% O2) or hypoxic (3% O2) conditions (f) and co-transduced with shVhl lentiviruses (g). h–o, Evaluation of [3H]leucine incorporation and the myosin heavy chain α/β mRNA expression ratio in NMCs treated with either phenylephrine or isoproterenol (h, l), HIF1αΔODD overexpression (i, m), hypoxia (j, n) or pVhl deletion (k, o), with or without shSf3b1 mediated Sf3b1 depletion. Obtained values are shown relative to incorporated radioactivity in control (shNS) NMCs (set as 1.0) (n = 4 per individual group). p, NMCs treated as in i were microscopically analysed for cell size using CellProfiler. Data represents 1 of 3 independent experiments with approximately 200 analysed cells per experiment. q, Lysates of NMCs treated as indicated, were assessed for protein expression of specified proteins by immunoblots. r, Evaluation of [3H]leucine incorporation of NMCs infected as indicated. Obtained values are shown relative to incorporated radioactivity in control (shNs/empty) NMCs (set as 1.0) (n = 3 per individual group). s–z, Assessment of extracellular acidification rate (ECAR), and oleic acid- and carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP)-induced oxygen consumption rate (OCR) in NMCs transduced and treated as indicated. Highlighted ECAR measurements were done at baseline. All data are compared to shNs-transduced NMCs. (n = 8 biological replicates in the group shNs + isoproterenol; n = 10 biological replicates in all other groups). Error bars are s.d. (a, b, h–o, r) or s.e.m. (p, s–z). *P < 0.05; **P < 0.01; ***P < 0.001. Two-tailed unpaired t-test (b, t, u, w–z). One-way ANOVA followed by Dunnett’s multiple comparison post-test (a, h–p, r).
Extended Data Figure 3 Influence of U2snRNP-complex members on KHK isoform expression and validation of KHK-C-mediated fructose uptake and metabolism.
a, NMCs were transduced with either shNs or shSf3b1 and transfected with codon optimized Sf3b1 or empty construct and expression of denoted proteins was assessed. b, NMCs were transduced with either shNs or shSf3b1 and transfected with codon-optimized Sf3b1 or empty construct, respectively. Exogenous Sf3b1-expression was evaluated by qRT–PCR. Three different primer pairs were used. Primer pair A detects both endogenous and exogenous codon-optimized Sf3b1 (top). Primer pair B recognizes Sf3b1 close to the shRNA binding site and is specific for endogenous Sf3b1 (left). Primer pair C is located in the codon-optimized region (right). Values were normalized to shNs/empty-vector-infected NMCs (n = 3 biological replicates per group). c, [3H]leucine incorporation normalized to cell number in NMCs treated as in b (n = 4 biological replicates per group). d, NMCs transduced with empty vector or human SF3A3 or SF3B3 were assessed for mRNA expression of Khk-A and Khk-C by qRT–PCR. Values were normalized to empty-vector-transduced NMCs (n = 3 biological replicates per group). e, NMCs transduced as in d were immunoblotted for indicated proteins. f, NMCs transduced as indicated were probed for mRNA expression of Khk-A or Khk-C by qRT–PCR. Values were normalized to shNs/empty-vector-transduced NMCs (n = 3 biological replicates). g, NMCs treated with either scrambled shRNA or two different shRNAs targeting either Sf3a3, Sf3b2 or Sf3b3 were co-transduced with empty vector or HIF1αΔODD. Protein expression of Khk-A/C or Khk-C specifically was evaluated by immunoblotting. h, Evaluation of [3H]leucine incorporation of NMCs infected as in d normalized to empty-vector-transduced NMCs (n = 4 biological replicates per group). i, Lysates of NMCs treated as indicated were immunoblotted for specified proteins. j, KHK-activity in lysates of NMCs transduced with a combination of lentiviruses expressing either shKhk targeting mouse Khk and human KHK-A or shKhk and human KHK-C or a scrambled shRNA control together with an empty vector. k, Evaluation of the kinetics of [13C]fructose-1-phosphate accumulation in NMCs treated as in i at the respective time points (n = 3 biological replicates). l, Glut5 mRNA expression in NMCs transduced as indicated. (n = 3 biological replicates per group). m, NMCs transduced as indicated were microscopically analysed for cell size. Data represents 1 of 3 experiments with approximately 50 analysed cells per experiment n, Khk-A- and Khk-C-specific shRNAs were evaluated for their potential to inhibit Khk-A and Khk-C isoform expression, respectively. NMCs were infected with shNs, shKhk-A or shKhk-C lentiviruses. Data are shown in relation to shNs NMCs (set to 1.0) (n = 3 biological replicates). Error bars are s.d. (b–d, f, h, k, l, n) or s.e.m. (m). *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA followed by Dunnett’s multiple comparison post-test (b–d, f, k, l, m, n).
a, log2 fold change of metabolites in NMCs overexpressing KHK-C and cultured with glucose or glucose and fructose compared to the corresponding control transduced NMCs (n = 4 biological replicates per group). b, Incorporation of [3H]fructose into RNA (top), DNA (middle) and protein (bottom) of NMCs treated as indicated (n = 4 biological replicates per group). c, Uptake of [14C]deoxyglucose in NMCs infected as in b at depicted time points (n = 4 biological replicates per group). d, [3H]leucine incorporation in NMCs transduced with shNs or shKhk and co-transduced with either empty overexpression vector or KHK-A or KHK-C respectively. NMCs were cultured in media with increasing fructose concentrations, under physiologic glucose amounts or with increasing glucose concentrations under physiologic fructose amounts. Data are presented relative to shNs/empty-vector-transduced NMCs at 5 mM glucose/0 μM fructose (n = 4 biological replicates). e, ADP/ATP ratios in NMCs transduced as indicated. (n = 4 biological replicates, data show 1 of 3 representative experiments). f, Immunofluorescence images of NMCs transduced with empty vector (upper panel) or HIF1αΔODD (middle panel) lentiviruses or exposed to hypoxia (lower panel) were additionally transduced as indicated. Prior to staining for sarcomeric α-actinin, Oil Red O and DAPI, NMCs were incubated with oleic acid. g, Quantification of lipid droplets/cell in NMCs of immunofluorescent images shown in f. h, Ratio of lipid droplets/cell area of NMCs shown in g. i–l, NMCs were infected as indicated and processed for oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements under basal conditions or after injection of OA or FCCP. Depicted are rates expressed as OCR to ECAR ratios (upper panels) or individual ECAR (for measurements at baseline) or OCR values (n = 8 biological replicates for HIF1αΔODD/shKhk-A; n = 10 biological replicates for all other groups). Error bars are s.d. (b–e) or s.e.m. (g–l). *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA followed by Dunnett’s multiple comparison post-test (b–e, g, h). Two-tailed unpaired t-test (i–l).
Extended Data Figure 5 Fructose and glucose uptake ex vivo and validation of AAV9-fl/fl-shSf3b1 viruses.
a, Ratio of left-ventricular weight to body weight of sham- or TAC-treated C57/Bl6J mice; (n = 7 per group). b, c, [3H]fructose (b) and [14C]deoxy-glucose (c) were co-applied by oral gavage and biodistribution was measured at 45 min post injection (n = 7 per group). d, Ratio of left-ventricular weight to tibia length (LV/TL) of sham- or 1K1C-treated C57/Bl6J wild-type mice (n = 6 for sham and n = 7 for 1K1C-treated mice). e, Fructose levels in probes from left ventricles of indicated mice as determined by a quantitative colorimetric assay, normalized to protein amount (n = 4 for sham and n = 5 for hearts of 1K1C-treated mice). f, g, Freshly isolated left-ventricular pieces of sham- or 1K1C-treated mice incubated in medium containing either [14C]fructose (f) or [14C]deoxyglucose (g) as indicated were processed for uptake measurements. Counts were normalized to tissue weight (n = 6 per time point and treatment). h–j, qPCR analyses of Glut5 mRNA expression in left ventricles of 1K1C- (h), TAC- (i) or isoproterenol-treated (j) mice. All values are presented in relation to Glut5 expression in shams (set as 1.0). n = 5 for sham-treated mice as controls for TAC-treated mice (n = 6), all other groups n = 7. k, l, Relative mRNA expression of GLUT5 and aldose reductase (AKR1B1) in left-ventricular biopsies of patients diagnosed with aortic stenosis (k) or HCM (l) in relation to healthy controls (set as 1.0) was analysed by qPCR (n = 6 for controls; n = 16 for HCM and n = 17 for patients with aortic stenosis). m, Integration of AAV9-fl/fl-shSf3b1#1 viruses in the myocardium of Mlc2-cre− or Mlc2v-cre+ mice was examined 12 weeks after intravenous injection by confocal microscopy (using 20× magnification) of left-ventricular cryoslices stained against GFP and sarcomeric α-actinin and counterstained for DAPI. n, Ventricular samples of Mlc2v-cre− or Mlc2-cre+ mice transduced with AAV9-fl/fl-shSf3b1#1 or AAV9-fl/fl-shSf3b1#2 viruses were assessed for expression of indicated proteins by immunoblotting. o, p, Ratio of left-ventricular weight to tibia length (o) and ejection fraction (%EF) (p) of sham- or TAC-treated Mlc2v-cre− and Mlc2v-cre+ mice injected with AAV9-fl/fl-shSf3b1#1 (number of mice per group is given in Extended Data Table 1). q, Lysates of hearts from sham- or TAC-operated Mlc2v-cre− and Mlc2v-cre+ mice transduced with AAV9-fl/fl-shSf3b1#1 viruses were processed for immunoblotting with antibodies as denoted. Error bars are s.e.m. (a–l, o, p). *P < 0.05; **P < 0.01; ***P < 0.001, two-tailed unpaired t-test (a–l, o, p).
Extended Data Figure 6 Pathologic growth is dependent on HIF1α-driven and SF3B1-mediated KHK-C expression.
a–f, Ratio of left-ventricular weight to tibia length (LV/TL) (a), mRNA expression of the hypertrophic markers Nppa (b), myosin heavy chain β (βMhc) (c) and myosin heavy chain α (αMhc) (d), ejection fraction (%EF) (e) and left-ventricular ATP content (f) of sham- or TAC-treated Khk-A+/+ and Khk-A−/− mice. g–l, Ratio of left-ventricular weight to tibia length (LV/TL) (g), mRNA expression of the hypertrophic markers Nppa (h), myosin heavy chain β (βMhc) (i) and myosin heavy chain α (αMhc) (j), ejection fraction (%EF) (k) and left-ventricular ATP content (l) of Khk-A/C+/+ and Khk-A/C−/− mice. m, n, Lysates from ventricles of sham- or TAC-operated Khk-A+/+ and Khk-A−/− mice (m) and Khk-A/C+/+ and Khk-A/C−/− mice (n) were processed for immunoblotting with antibodies as indicated. o–t, Assessment of hypertrophic marker gene expression (Nppa, βMhc and αMhc) in ventricles of TAC- vs sham-treated Khk-A−/− mice and their corresponding Khk-A+/+ controls (o–q) as well as Khk-A/C−/− mice and their corresponding Khk-A/C+/+ controls (r–t). u, v, Mean blood pressure (BP) in mmHg of Khk-A+/+ and Khk-A−/− (u) as well as Khk-A/C+/+ and Khk-A/C−/− mice (v) 8 weeks after sham or 1K1C treatment. For a–l, o–v, the number of mice per group is given in Extended Data Table 2. Error bars are s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed unpaired t-test.
Extended Data Figure 7 Model explaining how HIF1α activation of SF3B1-dependent splicing of KHK regulates fructose and glucose metabolism to promote cardiac hypertrophy in response to pathologic stress.
In this model, pathologic stress leads to increased expression of HIF1α and HIF1α-dependent activation of genes encoding glycolytic enzymes and the splicing factor SF3B1. SF3B1, in turn, assembles at the branch-point sequence upstream of exon 3C of KHK pre-mRNA leading to an inclusion of this exon and KHK-C protein production. This shift in isoform expression from KHK-A to KHK-C in response to HIF1α–SF3B1 pathway activation drives KHK-C-dependent fructose uptake via stimulation of GLUT5 expression, the conversion of fructose to fructose-1-phosphate (F1P) and contributes simultaneously to the activation of glucose uptake and metabolism through a yet-to-be-determined mechanism. The model holds that the unrestrained conversion of fructose to F1P by KHK-C limits ATP levels, thereby alleviating potential allosteric inhibition of phosphofructokinase (PFK) by ATP to maintain a high glycolytic flux. F1P is further metabolized to dehydroxyacetone phosphate (DHAP) and glyceraldehyde (GA). While DHAP serves as a precursor for glycerol synthesis, GA can be further converted to glyceraldehyde-3-phosphate (G3P), a key glycolytic intermediate. G3P can be channelled into the non-oxidative pentose phosphate pathway (PPP) supporting nucleic and amino acid biosynthesis. This metabolic constellation, created by the activation of the HIF1α–SF3B1–KHK-C axis, increases macromolecular biosynthetic capacity essential for hypertrophic growth, steatosis and cardiac dysfunction. HKII and F-1,6-BP denotes hexokinase II and fructose-1,6-bisphosphate, respectively.
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Mirtschink, P., Krishnan, J., Grimm, F. et al. HIF-driven SF3B1 induces KHK-C to enforce fructolysis and heart disease. Nature 522, 444–449 (2015). https://doi.org/10.1038/nature14508
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