Ufmylation Suppresses Unfolded Protein Response to Prevent Peripartum Cardiomyopathy

Figures

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  Figure 1. UFL1CKO Induces Cardiac Dysfunction After 1PP (A) Schematic of experimental design. Two-month-old control (CTL) and UFL1 knockout (UFL1CKO) female mice were used and mated with C57bl/6J mice for 3 terms of pregnancy followed by echocardiography 1 week after delivery. (B) Gross morphology of the mouse heart. (C) Heart weight to tibia length ratio. (D) Representative M-mode images. (E to L) Quantification of echocardiographic parameters of CTL and UFL1CKO mice before (CTL, n = 12; conditional knockout [CKO], n = 15) and after 1 pregnancy (1PP) (CTL, n = 16; CKO, n = 13). Left ventricular diastolic posterior wall thickness (LVPWd) or left ventricular systolic posterior wall thickness (LVPWs), left ventricular diastolic internal diameter (LVIDd) or left ventricular systolic internal diameter (LVIDs), left ventricular diastolic volume (LVVd) and left ventricular systolic volume (LVVs), left ventricular (LV) mass and LV ejection fractions (EFs) are shown. Data are presented as mean ± SEM. An unpaired Student's t-test was used in C, and 1-way analysis of variance with post hoc Tukey test was used in E to L. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. 2PP = 2 consecutive cycles of pregnancy;…

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  Figure 2. UFL1CKO Results in Heart Failure by 3PP (A) Gross morphology of the mouse hearts NP and 3PP. (B) Heart weight to tibia length. (C) M-mode images. (D to K) Quantification of echocardiographic parameters before and after 3 rounds of pregnancy. LVPWd or LVPWs, LVIDs or LVIDd, stroke volume (SV), LV EFs, and fractionation shortening (FS) are shown. Data are presented as mean ± SEM. An unpaired Student's t-test was used in B and a repeated measures 2-way analysis of variance with Tukey's post hoc test was used in D to K. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. Abbreviations as in Figure 1.

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  Figure 3. UFL1 Deficiency Triggers Pathological Cardiac Remodeling Following Pregnancy (A and B) Representative images (A) of wheat germ agglutinin (WGA) (green)-stained ventricular tissue sections showing cross-sectional cardiomyocyte area and the quantification (B). At least 500 cardiomyocytes per group were quantified, with a minimum of 150 cells per mouse heart and 3 mouse hearts per group. (C) Representative images of ventricular section stained by FastGreen and Sirius red. (D) Heatmap of indicated collagen genes that are significantly up-regulated in CKO+3PP hearts. Data were derived from RNA sequencing analysis of the indicated hearts. (E) Quantitative polymerase chain reaction analysis of indicated stress marker genes in the heart. (F) Representative images of transferase-mediated dUTP nick-end labeling (TUNEL)+ (red, arrowheads) cardiomyocytes in the hearts. The sections were counterstained with Phalloidin (green) and 4′,6-diamidino-2-phenylindole (blue) to mark cardiomyocytes and nuclei, respectively. Data are presented as mean ± SEM. Nested 1-way analysis of variance and 2-way analysis of variance followed by post hoc Tukey test were used in B and E, respectively. ∗P < 0.05;…

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  Figure 4. The Effects of ULF1CKO on Pregnancy-Induced Alterations in the Cardiac Transcriptome (A) Principal-component analysis showing gene expression patterns among different groups (n = 4 mice per group). (B) Volcano plot showing significantly down-regulated (blue) and up-regulated (red) genes in CTL+3PP, CKO+NP and CKO+3PP hearts, respectively (fold change [FC] >1.5 or <−1.5 and Padj < 0.05). (C) Bubble plot showing the enrichment of differentially expressed genes in the indicated hallmark gene set. (D) Venn diagram showing the enrichment of genes commonly dysregulated in CKO+NP (vs CTL+NP) and CKO+3PP (vs CTL+3PP) hearts in the indicated pathways. Abbreviations as in Figure 1.

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  Figure 5. Loss of UFL1 Induced ER Stress in the Hearts of Pregnant Mice (A) Enrichment plot of unfolded protein response gene set in indicated hearts. (B) Heatmap showing the expression levels of ER stress response genes in the hearts. (C) Enrichment plot showing over-representation of putative ATF4 target genes (GSE35681) in CKO+3PP hearts. (D) Venn diagram showing commonly up-regulated (up) and down-regulated (bottom) genes in CKO+3PP hearts and ATF4 overexpressed (ATF4OE) cardiomyocytes. (E) Quantitative real-time polymerase chain reaction analysis of indicated ER stress response genes in the hearts. (F and G) Western blot (F) and quantification (G) of indicated proteins using total heart lysates. Data are presented as mean ± SEM. Two-way analysis of variance followed by Tukey’s post hoc test was used in E and G. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. Abbreviations as in Figure 1.

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  Figure 6. UFL1 Deficiency Impaired Fatty Acid Oxidative Metabolism (A) Gene set enrichment analysis of hallmark oxidative phosphorylation gene set. (B) Heatmap of genes involved in oxidative phosphorylation in the hearts. (C) Quantitative polymerase chain reaction analysis of indicated mouse hearts. (D to F) Western blot (D) and quantification (E and F) of the mitochondrial proteins. (G to L) NRVCs were transfected with siRNA against luciferase (siLuci) or UFL1 (siUFL1) for 48 hours before additional analyses. (G and H) LipidTOX (green) staining (G) and quantification (H) of LipidTOX fluorescent intensity. NRVCs were incubated with oleic acid (100 mmol/L) for 24 hours before the analysis. Lipid droplets were stained with LipidTOX (green) staining. Cardiomyocytes and nuclei were counterstained with Phalloidin (red) and DAPI (blue), respectively. (I and J) Seahorse analysis of oxygen consumption rate (OCR) (I) and quantification (J) of basal and maximal respiration. (K) Representative confocal images of NRVC stained with Mitotracker (deep red), MitoSOX (green), and Hoechst (blue). (L) Quantification of the fluorescent intensity in (J). Over 50 cells per group were quantified. Data are…

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  Figure 7. UFL1 Deficiency Caused Hyperactivation of mTOR Signaling (A) Gene set enrichment analysis of the IGF1-mTOR pathway gene set. (B) Heatmap of several genes involved in the IGF1 pathway. (C) Western blot of indicated proteins in the hearts. (D to H) NRVCs were transfected with indicated siRNAs, followed by IGF1 (100 ng/mL) treatment for indicated times (D) or 24 hours (E to H). (D) Western blot of indicated proteins. (E and F) Puromycin incorporation assay. Cells were labeled with puromycin (0.5 μg/mL) treatment for 6 hours. Western blot (E) and quantification (F) of puromycin-incorporated proteins. (G and H) Confocal images (G) and the quantification (H) showing cell size. Data are presented as mean ± SEM. One-way analysis of variance with post hoc Tukey test was used in F and H. ∗P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Abbreviations as in Figure 1.