WWP1 Deficiency Alleviates Cardiac Remodeling Induced by Simulated Microgravity

Figures

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  FIGURE 1. WW domain-containing E3 ubiquitin protein ligase 1 expression changes in the hearts of mice and rhesus monkeys after simulated microgravity. (A) Representative western blotting analysis of WWP1 expression in cardiac extracts of adult mice following tail suspension (TS) for 6 weeks (n = 3 for each group). (B) Quantification of WWP1 protein levels of (A). (C) Immunohistochemistry for WWP1 (brown) in paraffin section from mouse hearts at 6 weeks after tail suspension. Scale bars, 200 μm. (D) Representative western blotting analysis of WWP1 expression in cardiac extracts of adult rhesus monkeys following bed rest (BR) for 6 weeks (Ctrl, n = 3; BR, n = 5). (E) Quantification of WWP1 protein levels of (D). (F) Immunohistochemistry for WWP1 (brown) in paraffin section from rhesus monkey hearts at 6 weeks after bed rest. Scale bars, 200 μm. Data represent the means ± SEM. *P < 0.05, **P < 0.01. WWP1, WW domain-containing E3 ubiquitin protein ligase 1; TS, tail suspension; BR, bed rest.

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  FIGURE 2. WW domain-containing E3 ubiquitin protein ligase 1 knock out alleviates simulated microgravity-induced cardiac function decline. (A,B) Echocardiographic assessment of ejection fraction (EF) and fractional shortening (FS) of WT and WWP1 knockout (KO) mice after 6 weeks of tail suspension. (C–F) Body weight, heart weight, tibia length, and the ratio of heart weight to tibia length of wild-type (WT) and WWP1 KO mice after 6 weeks of tail suspension. Data represent the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Ctrl, control; TS, tail suspension; WT, wild-type mice; KO, knockout.

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  FIGURE 3. The left ventricular structure of WT and WWP1 KO mice after tail suspension. (A–H) Quantitative analysis of the diastolic and systolic left ventricular posterior wall thickness (LVPWd and LVPWs), LV anterior wall thickness (LVAWd and LVAWs), LV internal diameter (LVIDd and LVIDs), and LV volume (LV Vold and LV Vols) from WT and WWP1 KO mice by echocardiography following tail suspension. Data represent the means ± SEM. *P < 0.05, **P < 0.01. Ctrl, control; TS, tail suspension; WT, wild-type mice; KO, knockout.

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  FIGURE 4. WW domain-containing E3 ubiquitin protein ligase 1 knockout protects against simulated microgravity induced-cardiac atrophy. (A) Hematoxylin and eosin (H&E)-stained sections of the hearts from WT and WWP1 KO mice after 6 weeks of tail suspension. Sections of the hearts are stained with Masson trichrome (MTT) to detect fibrosis (blue). Wheat germ agglutinin (WGA) staining is used to demarcate cell boundaries. (B) The cardiomyocyte cross-sectional area was measured from 7-μm-thick heart sections that have been stained with WGA by ImageJ software (NIH). Only myocytes that were round were included in the analysis. (C–E) The mRNA levels of BNP, Col1α1, and Col3α1 were analyzed by Q-PCR from WT and WWP1 KO mice after 6 weeks of tail suspension. The relative abundance of transcripts were quantified and normalized to Gapdh. Data represent the means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001. Col1α1, alpha-1 type I collagen; Col3α1, alpha-1 type III collagen; BNP, brain natriuretic peptide; Q-PCR, real-time quantitative polymerase chain reaction.

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  FIGURE 5. Disheveled segment polarity proteins 2 expression changes in the hearts of mice and rhesus monkeys after simulated microgravity. (A) Representative western blotting analysis of DVL2 expression in cardiac extracts of adult mice following tail suspension (TS) for 6 weeks (n = 3 for each group). (B) Quantification of DVL2 protein levels of (A). (C) Immunohistochemistry for DVL2 (brown) in paraffin section from mouse hearts at 6 weeks after tail suspension. Scale bars, 200 μm. (D) Representative western blotting analysis of DVL2 expression in cardiac extracts of adult rhesus monkeys following bed rest (BR) for 6 weeks (Ctrl, n = 3; BR, n = 5). (E) Quantification of DVL2 protein levels of (D). (F) Immunohistochemistry for DVL2 (brown) in a paraffin section from rhesus monkey hearts at 6 weeks after bed rest. (G) Pearson correlation coefficients between DVL2 and WWP1 protein levels in the heart of mice following tail suspension. (H) Pearson correlation coefficients between DVL2 and WWP1 protein levels in the heart of rhesus monkeys following bed rest. Scale bars, 200 μm. Data represent the means ± SEM. *P < 0.05, ***P < 0.001. DVL2, disheveled segment polarity protein 2; TS, tail…

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  FIGURE 6. WW domain-containing E3 ubiquitin protein ligase 1 knockout inhibits DVL2/CaMKII/HDAC4 signal in mice heart after simulated microgravity. (A) Representative western blots for WWP1, DVL2, and CaMKIIδ and phosphorylation at Thr287 and HDAC4 and phosphorylation at Ser246 in the hearts from WT and WWP1 KO mice after 6 weeks of tail suspension. GAPDH levels served as a loading control. (B–D) Quantification analysis of corresponding protein in (A). (E) Model of WWP1 function in simulated microgravity-induced cardiac remodeling signaling. Data represent the means ± SEM. ***P < 0.001. WWP1, WW-domain containing E3 ubiquitin protein ligase 1; DVL2, disheveled segment polarity protein 2; CaMKII, calcium/calmodulin-dependent protein kinase II; HDAC4, histone deacetylase 4; TS, tail suspension.