IRF1-mediated downregulation of PGC1α contributes to cardiorenal syndrome type 4

Figures

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  Fig. 1. Myocardial energy metabolism dysfunction and PGC1α downregulation are observed in the heart of CKD mice. a GO enrichment of the significantly changed cellular components of the heart from three pairs of sham and CKD mice. b KEGG pathway classification of differentially expressed genes (DEG) of the heart in sham and CKD mice. c Representative images of transmission electron microscope (TEM) observation of mitochondria of the heart from sham and CKD mice (n = 3 mice per group). Scale bar, 2 μm. Red arrows point to mitochondria. d Heatmap of DEG (mainly PGC1α and its target genes) of mice in a. The color intensity represents row Z-score. Red color indicates highly expressed, while blue indicates lowly expressed. e–g Relative mRNA expression of FAO genes (e), OXPHO genes (f), and PGC1α and its target genes (g) of the heart from sham and CKD mice. h Western blot analysis of PGC1α expression of the heart from sham and CKD mice. Microarray data were obtained from three mice per group. Data in a, b are shown as −LogP and were analyzed by Fisher’s exact test, adjusted by using Benjamini–Hochberg. Data in e–h are shown as mean ± SD and were analyzed by a two-tailed unpaired t-test (n = 3).…

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  Fig. 2. HP induces PGC1α reduction and mitochondrial energy metabolism dysfunction in vitro. a H9c2 cells were treated with urea (1.2 mg/ml), serum creatinine (Scr, 80 μg/ml), uric acid (UA, 80 μg/ml), parathyroid hormone (PTH, 90 ng/ml), trimethylamine-N-oxide (TMAO, 0.751 μg/ml), HP (84 μg/ml), FGF23 (20 ng/ml), β2-microglobulin (B2M, 20 μg/ml), indoxyl sulfate (IS, 125 μg/ml), p-cresyl sulfate (PCS, 47 μg/ml), and indole-3-acetic acid (IAA, 3.5 μg/ml), separately. mRNA was extracted for qPCR analysis of PGC1α expression. b, c qPCR and western blot analysis of PGC1α expression in H9c2 cells incubated with the serum of healthy donors or CKD patients for 24 h. d–g qPCR and representative western blot analysis of PGC1α expression in H9c2 cells treated with control, various doses of HP for 24 h or HP (8.4 mg/dl) for various durations. h–p Oxygen consumption rate (OCR) (h), relative ATP level (i), relative mRNA expression of FAO genes (j), relative mRNA expression of OXPHO genes (k), relative mRNA expression of glycolysis genes (l), relative mitochondrial DNA copy number (m), ROS production (n), and monomer and aggregate JC-1 (o flow cytometry; p representative images of laser scanning…

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  Fig. 3. HP induces PGC1α suppression and cardiac remodeling in CKD mice. a Serum phosphate level in sham, CKD, and HPD-fed CKD mice for 12 weeks. n = 10 mice per group. b–g qPCR analysis of PGC1α and its target genes expressions (b), western blot analysis of PGC1α expression (c), representative images of TEM observation of mitochondria (d). Red arrows point to mitochondria, relative ATP level (e), relative mRNA expression of FAO genes (f), and relative mRNA expression of OXPHO genes (g) in heart tissues from the mice in a. n = 10 (a, e), or n = 3 (b–d, f, g). Data are shown as mean ± SD and were analyzed by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Fig. 4. Restoration of PGC1α expression protects against HP-induced mitochondrial energy metabolism dysfunction and cardiac remodeling in both cardiac cells and CKD mice. a–d H9c2 cells were transfected with PGC1α overexpression plasmids or pretreated with Nam, and then treated with control or HP for representative western blot analysis of PGC1α expression (a) ROS production (b) relative mRNA expression of MCAD (FAO), Ndufa (OXPHO), and PFKP (glycolysis) (c), and relative ATP level (d). e Representative gross pathology (HE staining, upper panel. Scale bar, 1 mm) and WGA staining (lower panel. Scale bar, 10 μm) of heart sections from sham, HPD-fed CKD, Nam, and HPD-fed CKD mice intraperitoneally injected with Nam for 12 weeks. n = 10 mice per group. f Cross-sectional surface area of individual cardiac myocytes from the mice in the lower panel of (e). g–j Echocardiography of EF% (g), qPCR analysis of the mRNA expression of hypertrophic genes (h), relative ATP level (i), relative mRNA expression of metabolic genes (j) of heart lysates from the mice in (e). n = 3 (a–d, h, j), or n = 10 (e–g, i). Data are shown as mean ± SD and were analyzed by one-way ANOVA. *P < 0.05, **P < 0.01, ***P <…

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  Fig. 5. HP disturbs PGC1α expression and energy metabolism in an IRF1-dependent manner. a qPCR analysis of the expression of the predicted transcriptional factors in H9c2 cells treated with control or HP for 24 h. b, c qPCR and representative western blot analysis of IRF1 expression in H9c2 cells treated with control or various doses of HP for 24 h. d–g qPCR and representative western blot analysis of IRF1 expression in heart lysates from sham, CKD, and HPD-fed CKD mice for 12 weeks (d, e) or in H9c2 cells incubated with the serum of healthy donors or CKD patients for 24 h (f, g). h–l H9c2 cells were transfected with two pairs of siRNA against IRF1, and then treated with control or HP for 24 h. Cells were collected for detection of IRF1 and PGC1α protein expressions (h), PGC1α mRNA expression (i), ROS production (j), relative ATP level (k), and mRNA expression of the metabolic genes (l). m qPCR analysis of IRF1 and PGC1α expressions in H9c2 cells transfected with control plasmids (pCtrl) or IRF1 overexpression plasmids (pIRF1). n Representative western blot analysis of IRF1 and PGC1α expressions in H9c2 cells transfected with pCtrl, pIRF1, control siRNA (siCtrl), and two pairs of siRNA…

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  Fig. 6. IRF1 inhibits PGC1α expression via directly binding to its promoter region. a After being co-transfected with pRL-TK vector and pGL3-basic or recombinant reporter plasmids containing various fragments of PGC1α promoter region, H9c2 cells were treated with control or HP and then harvested for dual-luciferase reporter assay. The firefly luciferase activity was normalized against Renilla activity. b H9c2 cells were co-transfected with pRL-TK vector and pGL3-PGC1α-P3 or pGL3-PGC1α-M3 (containing the mutant bases of pGL3-PGC1α-P3 underlined in the sequencing results), treated with control or HP and harvested for luciferase assay. c, d H9c2 cells were treated with control or HP for 24 h and harvested for ChIP assay. IRF1 antibody was immunoprecipitated with chromatin DNA fragments, taking IgG as a negative control. The precipitated DNA was amplified by PCR c and qPCR d using primers that cover the PGC1α promoter region (−709 to −511), taking primers that cover the region (−1711 to −1598) as a negative control. e, f qPCR and representative western blot analysis of IRF1 and PGC1α expressions in cells treated with siPit1, siPit2, or both in the presence or absence of HP. g, h H9c2 cells…

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  Fig. 7. Klotho deficiency leads to hyperphosphatemia, LVH, and heart failure. a Serum phosphate levels of wild type (WT) and kl/kl mice. n = 9 mice per group. b The ratios of heart weight (HW) to body weight (BW), and HW to tibial length (TL) of mice in a. c, d Representative gross pathology (c HE staining, scale bar, 1 mm) and WGA staining (d scale bar, 10 μm) of heart sections from WT and kl/kl mice. e Cross-sectional surface area of individual cardiac myocytes from mice in d. f, g Echocardiography of LVIDd, LVPWd, and EF% in WT and kl/kl mice. h–k qPCR analysis of PGC1α and its target genes (h), western blot analysis of PGC1α (i), and IRF1 (j) expressions, representative images of TEM observation of mitochondria (k). Red arrows point to mitochondria. Scale bar, 2 μm, relative mRNA expression of metabolic genes (l), and relative ATP level m in the heart samples from WT and kl/kl mice. n = 9 (a, b, e, g, m), or n = 3 (h–l). Data are shown as mean ± SD and were analyzed by a two-tailed unpaired t-test (a, b, e, g–j, l–m). *P < 0.05, **P < 0.01, ***P < 0.001.

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  Fig. 8. Genetic knockdown of IRF1 protects against HPD-induced HF in CKD mice. a The ratios of HW to BW and HW to TL of WT, HPD-fed WT CKD, IRF1+/−, and HPD-fed IRF1+/− CKD mice for 12 weeks. n = 10 mice per group. b Representative gross pathology of heart sections (HE staining, upper panel, scale bar, 1 mm), WGA staining of the left ventricle of heart sections (lower panel, scale bar, 10 μm), and cross-sectional surface area of individual cardiac myocytes from mice in a. c, d Echocardiographic detection of LVIDd, LVPWd, and EF% in mice in a. e–h qPCR analysis of hypertrophic genes expression (e), western blot analysis of IRF1 and PGC1α expressions (f), relative ATP level (g), and relative mRNA expression of metabolic genes (h) of heart lysates from the mice in a. β-actin was taken as the loading control. n = 10 (a–d, g), or n = 3 (e, f, h). Data are shown as mean ± SD and were analyzed by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001 versus WT. #P < 0.05, ##P < 0.01, ###P < 0.001 versus CKD + HPD.

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  Fig. 9. A proposed model of the mechanism underlying myocardial energy metabolism remodeling and HF induced by HP. High phosphate (HP) enters cardiomyocytes via Pit1 and Pit2, and induces IRF1 expression through acetylating histone H3K9 in the IRF1 promoter region, during which histone acetyltransferases (HAT) are involved. HP-induced IRF1 inhibits PGC1α expression via directly binding to its promoter region (−632 to −612). PGC1α downregulation contributes to HP-induced mitochondrial dysfunction and myocardial energy metabolism remodeling. Restoration of PGC1α expression or knockdown of IRF1 protects against CKD-associated myocardial energy metabolism remodeling and HF.