RNA binding protein IGF2BP2 expression is induced by stress in the heart and mediates dilated cardiomyopathy

Figures

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  Fig. 1. Expression of IGF2BP paralogs and cardiac stress models. a Relative IGF2BP2 RNA expression was measured by RT-PCR in normal hearts at postnatal day 1 (P1), postnatal day 8 (P8), 3 weeks (3 W), and 6 weeks (6 W) of age. Each bar represents three biological repeats ±SEM). b Western blot analysis of IGF2BPs at the indicated time points. α/β Tubulin was used as a loading control. On the right, is the quantification of the normalized results. Two biological repeats are shown for each time point ±SEM. Each protein was probed on a different blot. c mRNA was extracted from the hearts of sham or TAC mice 10 weeks after the procedure and assayed by RT-PCR for IGF2BP2 expression (n = 3; p = 0.0015). d IGF2BP2 expression was detected by immunofluorescence (bottom row) on heart sections from sham-operated or TAC mice. Nuclei are stained with DAPI (top row). Scale bar is 250 µm. Statistical significance was determined by a two-tailed Student’s t-test. **p < 0.01.

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  Fig. 2. Activation of the conditional IGF2BP2 transgene in mouse hearts. a Kaplan–Meier survival curve of the control (MHC-tTA, n = 6) and IGF2BP2-expressing transgenic (MHC-tTA/IGF2BP2, n = 6) mice. Pregnant mothers were fed water without tetracycline (i.e., activation of the transgene occurred from E8.5). b Scatter plot/bar graph of the ratio of heart weight to body weight in control and MHC-tTA/IGF2BP2 mice. c Representative pictures of an MHC-tTA (left) heart and an MHC-tTA/IGF2BP2 heart (right) ±SEM. d H&E staining of a cross-section of an MHC-tTA (left) and MHC-tTA/IGF2BP2 heart (right). Scale bar is 500 µm. Asterisks indicate the sub-endocardium. e Kaplan–Meier survival curve of 8–10 weeks old transgenic mice (MHC-tTA/IGF2BP2, males n = 6, females n = 6; MHC-tTA, males n = 6, females n = 5). Tetracycline was withdrawn from drinking water on day 0. f Bar graph of the ratio of heart weight to body weight (HW/BW mg/g ± SEM) in adult MHC-tTA mice and MHC-tTA/IGF2BP2 mice at different time points after transgene induction (10d: MHC-tTA, n = 3; MHC-tTA/IGF2BP2, n = 5; 20d: MHC-tTA, n = 3; MHC-tTA/IGF2BP2, n = 4; 28d: MHC-tTA, n = 5; MHC-tTA/IGF2BP2, n = 3). g A representative picture of…

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  Fig. 3. Rescue experiment with full recovery. a Timeline of experimental design for the experiments described in (b–f): transgene on for 2 weeks and off for 12 weeks in adult mice. b and c Echocardiograms were performed on all the mice over the course of the experiment and analyzed for Ejection Fraction percentage (b) and fractional shortening percentage (c). d Scatter plot/bar graph of the ratio of heart weight to body weight (HW/BW) (mg/g) ±SEM in mice after 2 weeks of induction and 4 weeks after turning off the transgene. Below the graph are representative pictures of an MHC-tTA heart (left) next to an MHC-tTA/IGF2BP2 heart (right) (p = 0.0299) (MHC-tTA, n = 9; MHC-tTA/IGF2BP2, n = 5). e Wheat germ agglutinin staining of endocardial tissue adjacent to the papillary muscle from the hearts described in (d). Quantification of the area of 150 cardiomyocytes from each cohort is shown below. f Scatter plot/bar graph of the ratio of heart weight to body weight (HW/BW) (mg/g) ±SEM in mice after 2 weeks of induction and 12 weeks after turning off the transgene. Below the graph are representative pictures of two MHC-tTA (left) and four MHC -tTA/IGF2BP2 hearts (right) (MHC-tTA n = 5,…

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  Fig. 4. Rescue experiment with only partial recovery. a Timeline of experimental design for the experiments described in (b–f): transgene on for 2 weeks and 2 days and off for 12 weeks in adult mice. Echocardiograms were performed on all the mice over the course of the experiment and analyzed for Ejection Fraction percentage (b) and fractional shortening percentage (c). Three MHC-tTA controls were averaged together and are graphed in gray. The five MHC-tTA/IGF2BP2 mice are graphed individually and numbered I–V. d, e Picture of hearts at the end of the 2 weeks and 2 days transgene induction followed by 12 weeks off: d MHC-tTA control heart (left) and an MHC-tTA/IGF2BP2 heart (right) with normal EF% (mouse no. V) and e MHC-tTA control heart (left) and an MHC-tTA/IGF2BP2 heart (right) with impaired EF% (mouse no. III). f Trichrome staining of an MHC-tTA control and the different outcomes of the MHC-tTA/IGF2BP2 mice at the end of the experiment. Scale bar is 1 mm.

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  Fig. 5. Proteomics of uninduced and induced adult hearts. Protein extracts were prepared from MHC-tTA adult mice hearts (five samples, tTA controls) or from same-age siblings MHC-tTA/IGF2BP2 mice in which IGF2BP2 expression was activated for 15 days (four samples, IGF2BP2 OE) and analyzed by mass spectrometry. a Supervised hierarchical clustering of all 9 samples of differentially expressed proteins (with Z score, FDR = 0.05, S = 0.1; orange, upregulated; blue, downregulated). Top 20 Biological processes in GO analysis were ranked by −log10FDR (false discovery rate) of downregulated (b) and upregulated (c) proteins.

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  Fig. 6. EM images of the myocardium from transgenic mice. a Representative EM image of the LV myocardium of mice. Left panels, magnification was ×500, and right panels, ×1000. “m” indicates mitochondria. Note the absence of the H zone in both the MHC-tTA and MHC-tTA/IGF2BP2 hearts, indicating that both are in contraction. The double-headed arrows indicate the width of the myofibrils in the myocardium. Activation of IGF2BP2 expression leads to increased sarcomere length (b; p < 0.001) and decreased myofibril width (c; p < 0.001). IGF2BP2 activation also causes fractionalization of mitochondria, leading to an accumulation of smaller mitochondria (d) with a concomitant increase in their number per field (e; p = 0.0358). Accordingly, the average size per mitochondrion drops (f; p = 0.0037), although mitochondria still make up the same percentage of the area of the total field (g). h Mitochondrial DNA is reduced in hearts expressing IGF2BP2, as measured by PCR (p = 0.0466). b and c are aligned dot plots showing the mean value; e–h are scatter plots/bar graphs showing the mean and standard error of the mean. *p < 0.05, **p < 0.01 and ****p < 0.001.

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  Fig. 7. Expression of IGF2BP2 in human cardiomyopathies. Human pathological heart sections were stained with antibody against IGF2BP2. a Hypertrophic phenotype. b Dilatative cardiomyopathy. c Myocardial infarction. d Control heart. First row shows overview images at ×0.3 magnification, scale bar 5000 µm, the second row shows images at ×8 magnification, scale bar 100 µm, and the third row shows detail images at ×40 magnification, scale bar 20 µm. e The degree of IGF2BP2 expression was quantified as described in the “Methods” section. MI myocardial infarction, n = 13; and DCM, n = 20, p = 0.0404.