CITED4 Protects Against Adverse Remodeling in Response to Physiological and Pathological Stress

Abstract

Rationale: Cardiac CITED4 (CBP/p300-interacting transactivators with E [glutamic acid]/D [aspartic acid]-rich-carboxylterminal domain4) is induced by exercise and is sufficient to cause physiological hypertrophy and mitigate adverse ventricular remodeling after ischemic injury. However, the role of endogenous CITED4 in response to physiological or pathological stress is unknown.

Objective: To investigate the role of CITED4 in murine models of exercise and pressure overload.

Methods and results: We generated cardiomyocyte-specific CITED4 knockout mice (C4KO) and subjected them to an intensive swim exercise protocol as well as transverse aortic constriction (TAC). Echocardiography, Western blotting, qPCR, immunohistochemistry, immunofluorescence, and transcriptional profiling for mRNA and miRNA (microRNA) expression were performed. Cellular crosstalk was investigated in vitro. CITED4 deletion in cardiomyocytes did not affect baseline cardiac size or function in young adult mice. C4KO mice developed modest cardiac dysfunction and dilation in response to exercise. After TAC, C4KOs developed severe heart failure with left ventricular dilation, impaired cardiomyocyte growth accompanied by reduced mTOR (mammalian target of rapamycin) activity and maladaptive cardiac remodeling with increased apoptosis, autophagy, and impaired mitochondrial signaling. Interstitial fibrosis was markedly increased in C4KO hearts after TAC. RNAseq revealed induction of a profibrotic miRNA network. miR30d was decreased in C4KO hearts after TAC and mediated crosstalk between cardiomyocytes and fibroblasts to modulate fibrosis. miR30d inhibition was sufficient to increase cardiac dysfunction and fibrosis after TAC.

Conclusions: CITED4 protects against pathological cardiac remodeling by regulating mTOR activity and a network of miRNAs mediating cardiomyocyte to fibroblast crosstalk. Our findings highlight the importance of CITED4 in response to both physiological and pathological stimuli.

Keywords: exercise; extracellular matrix; heart failure; signal transduction.

Figure 1:. Cardiomyocyte specific CITED4 knockout does not affect baseline cardiac mass or function in young adult mice.

A, Baseline Fractional Shortening (%FS) in C4KO compared to F/F Ctr mice in 8-12 week old mice (n=4 F/F Ctr, 3 C4KO, p=0.54). B, Left ventricular mass index (LVMI) for C4KO and control animals (n=4 F/F Ctr, 3 C4KO, p=0.64). C, Heart weight to tibia length ratios (HW/TL) in C4KO and F/F Ctr animals (n= 3 mice per group, p=0.56). D, Lung weight to tibia length ratios (LW/TL) in C4KO and F/F Ctr animals (n=3 mice per group), p=0.25). E, Cross sectional area (CSA) of C4KO relative to F/F Ctr cardiomyocytes at baseline (n=4 F/F Ctr, 3 C4KO, p=0.057). F, Micrographs of cardiac sections stained with Masson Trichrome Staining used to measure cross sectional area in E, scale bar = 75μm, images were chosen to represent average of both groups. For all graphs significance was determined by Student’s t-test, *p<0.05.

Figure 2:. Cardiomyocyte specific deletion of CITED4 causes maladaptive remodeling and a functional deficit in response to endurance exercise.

A, Fractional Shortening (%FS) in C4KO compared to F/F Ctr mice after swimming exercise (n=5 F/F Ctr, 6 C4KO, p=0.005). B, Heart weight to tibia length ratios (HW/TL) in C4KO and F/F Ctr animals (n=5 F/F Ctr, 6 C4KO, p=0.29) after swimming exercise. C, Cross sectional area (CSA) of C4KO relative to F/F Ctr cardiomyocytes at baseline (n=3 F/F Ctr, 4 C4KO, p=0.015). D, Left ventricular internal dimension in end-diastole (LVIDd, p=0.043) and end-systole (LVIDs, p=0.049) as well as relative wall thickness (RWT, p=0.097) assessed by echocardiography in C4KO and control animals after swimming exercise (n=5 F/F Ctr, 6 C4KO). E, qPCR analysis of fibrosis-related genes in C4KO relative to F/F Ctr hearts after swimming exercise (Col3a1 p<0.001, Col5a1 p=0.008, CTGF p=0.017. F, qPCR analysis related to physiological and pathological remodeling (bMHC, ANP, BNP p=0.002), autophagy (Beclin1 p=0.023) and mitochondrial pathways (PGC1a, NDUFs1, NDUFv2) and mTOR regulators (DDIT4 p=0.027, DDIT4L) in C4KO relative to F/F Ctr hearts after swimming exercise (n=4 F/F Ctr, 5 C4KO). For all graphs significance was determined by Student’s t-test, *p<0.05.

Figure 3:. Cardiomyocyte specific CITED4 knockout mice develop accelerated heart failure after transverse aortic constriction (TAC).

A, qPCR analysis of CITED4 mRNA in hearts of control mice at 1 week (n=3) and 8 weeks (n=5) after TAC and at baseline (n=4) (p<0.035 baseline vs. 1 week, p<0.028 1 week vs. 8 weeks, p=0.874 baseline vs. 8 weeks, one-way ANOVA with Sidak’s multiple comparisons test). B, Echocardiographic images from F/F Ctr and C4KO 8 weeks after TAC, images were chosen to represent average of both groups. C, Assessment of ventricular function by echocardiography in C4KO compared to F/F Ctr mice at indicated time points before and after TAC surgery (n=6 F/F Ctr, 6 C4KO, p=.999, 0.050, 0.064, 0.002, <0.001, respectively). D, Left ventricular dimensions in end-diastole (LVIDd, p=0.999, 0.245, 0.009, 0.025, 0.282, respectively) and E, end-systole (LVIDs) at indicated time points before and after TAC surgery (n=6 F/F Ctr, 6 C4KO, p=0.999, 0.426, 0.076, 0.017, <0.0001, respectively). F, Relative wall thickness assessed by echocardiography in C4KO and control animals at indicated time points before and after TAC surgery (n=6 F/F Ctr, 6 C4KO, p=0.998, 0.026, 0.391, 0.049, <0.607, respectively). For all graphs in C-F significance was determined by repeated-measures two-way ANOVA and Sidak’s multiple comparisons test, *p<0.05.

Figure 4:. Cardiomyocyte specific CITED4 knockout mice develop ventricular dilation and maladaptive remodeling in response to pressure overload.

A, Heart weight to tibia length ratios (HW/TL) in C4KO and F/F Ctr animals at endpoint after TAC surgery (n=6 F/F Ctr, 7 C4KO, p=0.046). B, Lung weight to tibia length ratios (LW/TL) in C4KO and F/F Ctr animals at endpoint after TAC surgery (n=6 F/F Ctr, 7 C4KO, p=0.049). C, Cross sectional area measurement from C4KO compared to F/F Ctr hearts at endpoint after TAC surgery (n=4 F/F Ctr, 7 C4KO, >200 cells per section, p<0.001). D, Quantification of fibrosis measured as percentage of total myocardial area in C4KO compared to F/F Ctr hearts at the endpoint after TAC surgery (n=5 F/F Ctr, 4 C4KO, p=0.017). E, Micrographs of cardiac sections stained with Masson Trichrome Staining to label fibrotic tissue one week after TAC surgery, scale bar = 1mm, images were chosen to represent differences of fibrosis pattern in both groups. F, Images of TUNEL staining in C4KO compared to F/F Ctr hearts one week after TAC surgery. Sections were stained with Wheat Germ Agglutinin (WGA) to mark cellular membranes (green), TUNEL (red), and DAPI (blue), scale bar=100μm, images were chosen to represent TUNEL positive areas in both groups. G, Quantification of TUNEL-positive cardiomyocytes in C4KO relative to F/F Ctr hearts one week after TAC surgery (n=5 F/F Ctr, 4 C4KO, p=0.044). For all graphs significance was determined by Student’s t-test, *p<0.05 unless indicated otherwise.

Figure 5:. mTOR signaling is impaired in C4KO hearts after pressure overload.

A, Western Blot of protein isolated from heart lysates one week after TAC surgery comparing AKT, TSC2, S6- and 4EBP-protein-phosphorylation and Beclin1 and LC3 protein expression in C4KO hearts with F/F Ctr hearts. GAPDH served as protein loading control. B, Quantification of AKT (p=0.008), T1462-TSC2 (p=0.024), S939-TSC2 (p=0.047), S1387-TSC2 (p=0.063), pS6 (p=0.026) and p4EBP (p=0.012) protein-phosphorylation and Beclin1 (p=0.003) and LC3 A/B (p=0.087) protein expression in C4KO hearts relative to F/F Ctr hearts one week after TAC (n=6 F/F Ctr, 6 C4KO, *p<0.05, Student’s t-test). C, qPCR analysis of mTORC1 inhibitors REDD1 (DDIT4) and REDD2 (DDIT4L) in C4KO relative to F/F Ctr hearts one week after TAC surgery (n=4/group, *p=0.032 and 0.023, respectively, Student’s t-test). D, qPCR analysis of REDD1 (DDIT4) and REDD2 (DDIT4L) mRNA expression in isolated ventricular myocytes from F/F Ctr and C4KO hearts at baseline (n=5/group, p=0.029 REDD1 and 0.799 for REDD2) and after Phenylephrine (PE) stimulation (n=4 F/F Ctr, 3 C4KO, *p=0.002 for REDD1 −PE, 0.035 +PE, ns for REDD2, *p<0.05 one-way ANOVA and Sidak’s post test).

Figure 6:. Gene expression array reveals impaired mitochondrial pathways in C4KO hearts after pressure overload.

A, Heatmap depicts differentially expressed mRNA (applying the combined criteria of p≤0.005 and fold change ≥1.5) in C4KO and F/F Ctr heart samples (n=4/group) one week after TAC surgery. The dendrogram was constructed using the Manhattan-Ward clustering algorithm. B, Bar-plot of the top 10 most significantly enriched pathways, Gene Set Enrichment Analysis (GSEA) using Gene Ontology database. Bars depict the normalized enrichment score of pathway up- or downregulation in C4KO hearts relative to F/F Ctr hearts one week after TAC surgery (n=4/group). C, qPCR for validation of reduced PGC1α, NDUFs1, and NDUFv2 mRNA expression (n=4 F/F Ctr, 4 C4KO, p=0.001, 0.039, 0.024, respectively), Student’s t-test, *p<0.05.

Figure 7:. Fibrosis-related pathways are dysregulated in C4KO hearts after pressure overload.

A, Differentially expressed, fibrosis-related (annotated and/or published) miRNAs in C4KO compared to F/F Ctr hearts one week after TAC surgery assessed by miRNASeq profiling (n= 5/group). B, qPCR validation of fibrosis-related genes (mRNA) inversely regulated by differentially expressed miRNAs (A) in C4KO relative to F/F Ctr hearts after TAC surgery (n=4/group, p=0.041, 0.033, 0.021, 0.045, 0.048, 0.036, respectively, *p<0.05, Student’s t-test). C, qPCR validation of miR-30d expression in C4KO relative to F/F Ctr hearts one week after TAC surgery (n=8/group, p=0.045) and in hearts from inducible CITED4 mice relative to tTA Ctr hearts (n=4/group, p=0.029), *p<0.05, Student’s t-test. D, Neonatal rat ventricular myocytes (NRVM) were treated with negative control (Ctr) and CITED4 (C4) siRNA. C4 siRNA treatment lead to a reproducible decrease in CITED4 mRNA expression (p=0.002) and caused a decrease in miR30d expression (p=0.006) (n=4 individual experiments, *p<0.05, Student’s t-test). E, CTGF (*p<0.001, ^#p<0.001, Ctr miR vs. C4 siRNA p=0.002), Col1a1 (*p<0.001, Ctr miR vs. C4 siRNA p<0.001, p=0.039 Ctr miR vs. C4+miR30d, miR30d vs. C4 siRNA p=0.037), Acta2 (*p<0.001, ^#p<0.001, miR30d vs. C4 siRNA p<0.001) and Postn (*p=0.010, ^#p=0.033, miR30d vs. C4 siRNA p=0.014) mRNA expression evaluated in mouse embryonic fibroblasts (MEF) that were either treated with conditioned media from Ctr siRNA, Ctr miRNA mimic or C4 siRNA, miR30d mimic, or C4 siRNA in addition to miR30d mimic NRVMs (n=3 individual experiments, *p<0.05 vs. Ctr siRNA and Ctr miR, ^#p<0.05 vs. C4 siRNA, one-way ANOVA and Sidak’s post test).

Figure 8:. LNA mediated miR30d depletion leads to rapid cardiac failure and fibrosis in response to pressure overload.

A, Confirmation of successful locked nucleic acid (LNA) mediated miR30d depletion, relative miR30d expression in heart lysates from LNA-anti-miR30d and scramble (Scr) control LNA treated mice (n=4 Scr-LNA, 7 LNA-anti-miR30d, Student’s t-test, p<0.001, *p<0.05). B, Assessment of ventricular function by echocardiography in LNA-anti-miR30d compared to Scr-LNA treated mice at indicated time points before and after TAC surgery (n=6 Scr-LNA, 6 LNA-anti-miR30d, p=0.999, 0.999, 0.035, 0.033, respectively, repeated-measures two-way ANOVA with Sidak’s post test for multiple comparisons, *p<0.05). C, Left ventricular dimensions in end-diastole (LVIDd) at indicated time points before and after TAC surgery (n=6 Scr-LNA, 6 LNA-anti-miR30d, p=0.999, 0.922, 0.037, 0.034, respectively, repeated-measures two-way ANOVA with Sidak’s post test for multiple comparisons, *p<0.05). D, Heart weight to tibia length ratios (HW/TL) in LNA-anti-miR30d and Scr-LNA treated animals 4 weeks after TAC surgery (n=6 Scr-LNA, 9 LNA-anti-miR30d, Student’s t-test, p=0.010, *p<0.05). E, Lung weight to tibia length ratios (LW/TL) in LNA-anti-miR30d and Scr-LNA treated animals 4 weeks after TAC surgery (n=6 Scr-LNA, 9 LNA-anti-miR30d, Student’s t-test, p=0.036, *p<0.05). F, Quantification of fibrosis measured as percentage of total myocardial area in LNA-anti-miR30d compared to Scr-LNA treated hearts 4 weeks after TAC surgery (n=6 Scr-LNA, 5 LNA-anti-miR30d, Student’s t-test, p=0.047, *p<0.05) G, Micrographs of cardiac sections stained with Masson Trichrome Staining to label fibrotic tissue 4 weeks after TAC surgery, scale bar=100μm, images were chosen to represent differences of fibrosis pattern in both groups.