Role of a TRIM72 ADP-ribosylation cycle in myocardial injury and membrane repair
Hiroko Ishiwata-Endo, Jiro Kato, Akihiko Tonouchi, Youn Wook Chung, Junhui Sun, Linda A. Stevens, Jianfeng Zhu, Angel M. Aponte, Danielle A. Springer, Hong San, Kazuyo Takeda, Zu-Xi Yu, Victoria Hoffmann, Elizabeth Murphy, Joel Moss
Hiroko Ishiwata-Endo, Jiro Kato, Akihiko Tonouchi, Youn Wook Chung, Junhui Sun, Linda A. Stevens, Jianfeng Zhu, Angel M. Aponte, Danielle A. Springer, Hong San, Kazuyo Takeda, Zu-Xi Yu, Victoria Hoffmann, Elizabeth Murphy, Joel Moss
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Research Article Cardiology Muscle biology

Role of a TRIM72 ADP-ribosylation cycle in myocardial injury and membrane repair

Abstract

Mono-ADP-ribosylation of an (arginine) protein catalyzed by ADP-ribosyltransferase 1 (ART1) — i.e., transfer of ADP-ribose from NAD to arginine — is reversed by ADP-ribosylarginine hydrolase 1 (ARH1) cleavage of the ADP-ribose–arginine bond. ARH1-deficient mice developed cardiomyopathy with myocardial fibrosis, decreased myocardial function under dobutamine stress, and increased susceptibility to ischemia/reperfusion injury. The membrane repair protein TRIM72 was identified as a substrate for ART1 and ARH1; ADP-ribosylated TRIM72 levels were greater in ARH1-deficient mice following ischemia/reperfusion injury. To understand better the role of TRIM72 and ADP-ribosylation, we used C2C12 myocytes. ARH1 knockdown in C2C12 myocytes increased ADP-ribosylation of TRIM72 and delayed wound healing in a scratch assay. Mutant TRIM72 (R207K, R260K) that is not ADP-ribosylated interfered with assembly of TRIM72 repair complexes at a site of laser-induced injury. The regulatory enzymes ART1 and ARH1 and their substrate TRIM72 were found in multiple complexes, which were coimmunoprecipitated from mouse heart lysates. In addition, the mono-ADP-ribosylation inhibitors vitamin K1 and novobiocin inhibited oligomerization of TRIM72, the mechanism by which TRIM72 is recruited to the site of injury. We propose that a mono-ADP-ribosylation cycle involving recruitment of TRIM72 and other regulatory factors to sites of membrane damage is critical for membrane repair and wound healing following myocardial injury.

Authors

Hiroko Ishiwata-Endo, Jiro Kato, Akihiko Tonouchi, Youn Wook Chung, Junhui Sun, Linda A. Stevens, Jianfeng Zhu, Angel M. Aponte, Danielle A. Springer, Hong San, Kazuyo Takeda, Zu-Xi Yu, Victoria Hoffmann, Elizabeth Murphy, Joel Moss

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Figure 5

Depletion of ARH1, ART1, and TRIM72 in C2C12 cells delayed wound healing and membrane repair.

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Depletion of ARH1, ART1, and TRIM72 in C2C12 cells delayed wound healing...
(A) Western blot analysis of C2C12 cell lysates after TRIM72 (shTRIM72), ARH1 (shARH1), ART1 (shART1), and ARH1 plus ART1 (shARH1/ART1) shRNA transformation, demonstrating decreased protein expression compared with control scrambled shRNA (shCont.) transfectant. (B and C) Laser wounding assays were performed with C2C12 cells transformed with TRIM72-GFP in control scrambled shRNA (n = 35), ARH1 shRNA (n = 34), ART1 shRNA (n = 31), TRIM72 shRNA (n = 35), or double knockdown for shART1 and shARH1 (n = 31) with or without overexpression with WT TRIM72-GFP or mutant TRIM72(R207K,R260K)-GFP (n = 37) (B) (Supplemental Videos 5–10). (C) Measurements of GFP intensity at injury sites. (D and E) Scratch wound healing assays were performed with cells transformed with shCont., shARH1, shART1, shTRIM72, or shART1/ARH1 knockdown cells with or without overexpression with mutant TRIM72(R207K,R260K)-GFP or WT TRIM72–GFP. Scale bar: 1 mm. (E) Quantification of the area covered by cells at each time is shown as percentage of time 0 wound area (n = 12). (F) Western blots are shown for detection of ADP-ribosylated TRIM72 coimmunoprecipitated with active or inactive Af1521 macrodomain–GST from C2C12 myotube lysates with scratching with needles. The amount of ADP-ribosylated TRIM72 was increased after scratching of C2C12 cells transformed with control shRNA or ARH1 shRNA. In addition, in scratched C2C12 myocytes, Af1521 macrodomain–GST pull-down revealed that WT TRIM72-GFP, but not mutant-TRIM72(R207K,R260K)-GFP, was ADP-ribosylated. ADPr-TRIM72 coimmunoprecipitated with Af1521 macrodomain–GST was detected by Western blotting (WB) with anti-TRIM72 antibody. Data shown are representative of 3 experiments. Data are mean ± SEM. Open symbols are values significantly different (P < 0.05) from those of shCont. cells by 2-way ANOVA, followed by Bonferroni’s post hoc tests.