Herpud1 negatively regulates pathological cardiac hypertrophy by inducing IP3 receptor degradation

Figures

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  Figure 1. Expression of Herpud1 protein in mouse tissues. Representative Western blot of Herpud1 protein levels in heart, brain, skeletal muscle (extensor digitorum longus), and liver samples from wild-type (WT) and Herpud1-knockout (KO) mice (n = 3). *Non-specific band.

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  Figure 2. Herpud1-KO mice develop cardiac hypertrophy. (a) Four-chamber image, 40X, with hematoxylin and eosin staining (n = 3). (b) Representative images (400X) of paraffin embedded cardiac tissue stained with wheat germ agglutinin (WGA) and quantification of cardiomyocyte cross-sectional area (n = 6). (c) Heart weight/body weight (mg g−1, n = 12). (d) Heart weight/tibia length (mg cm−1, n = 12). (e) Left and right ventricular plus septum weight/tibia length (mg cm−1, n = 6). (f) Left and right atrial weight/tibia length−1 (mg cm−1, n = 6) in wild-type (WT) and Herpud1-knockout (Herpud1-KO) 10-to-12-week-old mice. Mean ± SEM, analyzed using a t-test. *p < 0.05 vs. WT.

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  Figure 3. Herpud1-KO mice develop myocardial dysfunction. (a) Representative long-axis echocardiography images, 2D and M-mode, of the left ventricle. LV: Left ventricle, Pap: papillary muscle. (b) % Fractional shortening (n = 12). (c) % Ejection fraction (n = 12). (d) Stroke volume (n = 12) in wild-type (WT) and Herpud1-knockout (Herpud1 KO) 10-to-12-week-old mice. Mean ± SEM, analyzed using a t-test. *p < 0.05 vs. WT.

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  Figure 4. Herpud1 siRNA triggers cardiomyocyte hypertrophy. (a) Representative Western blot of Herpud1 protein levels in isolated neonatal (Neo) and adult (Adu) cardiomyocytes (Cardio) and neonatal fibroblasts (Fibro) cultured under basal conditions or treated with tunicamycin for 4 h (Tn, 1.2 mM). Ponceau red shows the loading of each sample (n = 3). (b) Representative images (400X) of fixed NRVM stained with the fluorescent probe rhodamine-phalloidin after incubation with unrelated siRNA (UNR, 200 nM) or anti-Herpud1 siRNA (siHerpud1 #1) (200 nM) for 48 h (n = 3). (c) White arrows show analyzed sarcomeres in the fluorescent profiles of sarcomeres from the previous images. (d) Results for area, perimeter, and percentage of sarcomerized cells for fixed NRVM stained with rhodamine-phalloidin after incubation with unrelated siRNA (UNR) or anti-Herpud1 siRNA (siHerpud1 #1) for 48 h (n = 3). (e) Representative Western blots for Herpud1 (n = 6), β-MHC (n = 6), and Rcan1.4 (n = 4) for NRVM incubated with unrelated siRNA (UNR) or anti- Herpud1 siRNA (siHerpud1#2) for 48 h. Graphs represent the measurements for each protein using β-tubulin as the loading control. Mean ± SEM, analyzed using a…

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  Figure 5. IP3R protein and mRNA levels in cardiomyocytes treated with Herpud1 siRNA and in hearts from Herpud1-KO mice. (a) Representative Western blot of IP3R protein levels in Left: control (UNR) and Herpud1-knockdown NRVM (siHerpud1 #2, 48 h) (n = 6) and Right: samples from wild-type (WT) and Herpud1-knockout (Herpud1 KO) mice (n = 3). (b) Graphs show the relative expression of IP3R1, IP3R2, and IP3R3 mRNA in control (UNR) and Herpud1-knockdown NRVM (siHerpud1 #2, for 48 h) (n = 4) and IP3R2 mRNA in cardiac samples from wild-type (WT) and Herpud1-knockout (Herpud1 KO) 10-to-12-week-old mice (n = 3). Pabpn1 was used as a housekeeping messenger in isolated NRVM, and the 18 S ribosomal subunit was used in the cardiac tissue samples. Mean ± SEM, analyzed using a t-test. *p < 0.05 vs. UNR.

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  Figure 6. Effect of Herpud1 siRNA on polyubiquitin levels and cytosolic and nuclear Ca2+ levels in cultured NRVM. (a) Representative Western blots for lysine 48-linked polyubiquitin (Poly-Ub) in control (UNR) and Herpud1-knockdown NRVM (siHerpud1 #2, 200 nM, 48 h) in the presence or absence of the proteasome inhibitor MG-132 (4 μM, 24 h). Graphs correspond to the quantification of each lane, using β-tubulin as a loading control (n = 6). (b) Graph of cytosolic Ca2+ concentration kinetics and quantification of the area under the curve (n = 3). (c) Graph of nuclear Ca2+ kinetics and quantification of the area under the curve (n = 3). Both (b) and (c) correspond to control (UNR) and Herpud1-knockdown NRVM (siHerpud1 #2, 48 h). (d) Graph of cytosolic Ca2+ concentration kinetics and quantification of the area under the curve, in control (UNR) and Herpud1-knockdown NRVM (siHerpud1 #2, 48 h) in the absence or presence of xestospongin C (XeC, 100 μM, 30 min) (n = 3). The black arrow shows the time of addition of histamine (100 mM). Mean ± SEM analyzed using a t-test or one-way ANOVA followed by Bonferroni’s post-test. *p < 0.05 vs. UNR, † p < 0.05 vs. UNR + MG-132; # p < 0.05 vs. same condition…

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  Figure 7. Proposed model for hypertrophy induced by Herpud1 silencing. Herpud1 plays a role in the regulation of Ca2+ levels in cardiomyocytes, possibly by modulating IP3R degradation. Reduction in Herpud1 protein levels allows for activation of pro-hypertrophic signaling cascades such as the calcineurin/NFAT pathway. This new evidence suggests an alternative model for the development of pathological cardiac hypertrophy and a novel putative pharmacological treatment target.