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 1

Impaired cardiac function with myocardial fibrosis in ARH1-KO mice.

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Impaired cardiac function with myocardial fibrosis in ARH1-KO mice. (A a...
(A and B) Representative images of animals treated as described in Methods by echocardiography in M-mode from healthy WT (A) and cardiomyopathy ARH1-KO (B) mice (see also Supplemental Videos 1–4, and original images in the Supplemental Material). (C and D) Pictures of cross section of healthy WT mouse heart (C) and 8-month-old ARH1-KO heart with cardiomyopathy (D). White asterisks indicate left ventricle (scale bars: 1 mm). (E–H) Masson’s trichrome staining of myocardium from WT (E and G) and ARH1-KO (F and H) mice (scale bars: 200 μm). Histochemical staining for fibrosis shows collagen deposition in blue. (I) Frequency of myocardial fibrosis in WT (0.4%, 1 of 243 males; 0.8%, 2 of 247 females), heterozygous (HET, 0.8%, 2 of 254 males; 0.5%, 2 of 399 females) and ARH1-KO (22.2%, 48 of 216 males; 2.2%, 7 of 313 females) mice. *P < 0.05, ****P < 0.0001, significant difference compared with WT of same sex by 1-sample t test. (J–U) Dobutamine stress test was performed in WT males, n = 15, 37.1 ± 0.9 weeks; KO males, n = 15, 38.3 ± 3.9 weeks; WT females, n = 12, 39.2 ± 5.6 weeks; KO females, n = 13, 38.8 ± 3.5 weeks. Measurements of ejection fraction (EF; J and K); fractional shortening (FS; L and M), left ventricular internal dimension at systole (LVIDs; N and O) and diastole (LVIDd; P and Q), stroke volume (SV; R and S), and heart rate (T and U). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. WT of same sex by 2-way ANOVA, followed by Bonferroni’s post hoc tests.