A knock-in mutation at cysteine 144 of TRIM72 is cardioprotective and reduces myocardial TRIM72 release

Abstract

TRIM72 is a membrane repair protein that protects against ischemia reperfusion (I/R) injury. We previously identified Cys¹⁴⁴ (C144) on TRIM72 as a site of S-nitrosylation. To study the importance of C144, we generated a knock-in mouse with C144 mutated to a serine (TRIM72 C144S). We subjected ex vivo perfused mouse hearts to 20 min of ischemia followed by 90 min of reperfusion and observed less injury in TRIM72 C144S compared to WT hearts. Infarct size was smaller (54 vs 27% infarct size) and cardiac functional recovery (37 vs 62% RPP) was higher for the TRIM72 C144S mouse hearts. We also demonstrated that TRIM72 C144S hearts were protected against I/R injury using an in vivo LAD occlusion model. As TRIM72 has been reported to be released from muscle we tested whether C144 is involved in TRIM72 release. After I/R there was significantly less TRIM72 in the perfusate normalized to total released protein from the TRIM72 C144S compared to WT hearts, suggesting that C144 of TRIM72 regulates myocardial TRIM72 release during I/R injury. In addition to TRIM72's protective role in I/R injury, TRIM72 has also been implicated in cardiac hypertrophy and insulin resistance, and secreted TRIM72 has recently been shown to impair insulin sensitivity. However, insulin sensitivity (measured by glucose and insulin tolerance) of TRIM72 C144S mice was not impaired. Further, whole body metabolism, as measured using metabolic cages, was not different in WT vs TRIM72 C144S mice and we did not observe enhanced cardiac hypertrophy in the TRIM72 C144S mice. In agreement, protein levels of the TRIM72 ubiquitination targets insulin receptor β, IRS1, and focal adhesion kinase were similar between WT and TRIM72 C144S hearts. Overall, these data indicate that mutation of TRIM72 C144 is protective during I/R and reduces myocardial TRIM72 release without impairing insulin sensitivity or enhancing the development of hypertrophy.

Figure 1.. TRIM72 C144S protects against myocardial ischemia/reperfusion injury.

Hearts were Langendorff perfused and subjected to a A. 20 min ischemia and 90 min reperfusion protocol. B. Cardiac functional recovery and C. infarct size were measured. Values are mean ± SEM (WT n=5; TRIM72 C144S n=5); T-tests were performed to assess significance, * p<0.05 compared to WT

Figure 2.. TRIM72 C144S is protective in an in vivo model of LAD occlusion.

Mice were subjected to A. LAD occlusion and ischemia for 45 min followed by 24 hr reperfusion. B. Ejection fraction, C. infarct size, and D. area at risk (AAR) were measured. Values are mean ± SEM (WT n=12; TRIM72 C144S n=8); T-tests were performed to assess significance (*p<0.05 compared to WT).

Figure 3.. TRIM72 release is reduced in TRIM72 C144S in response to I/R injury.

Hearts were subjected to A. 20 min ischemia and 10 min reperfusion and perfusate was collected during the 10 min reperfusion period. B. TRIM72 protein was measured by western blot in the heart perfusate and normalized to the Ponceau S stain for total protein. Values are mean ± SEM (WT n=4; TRIM72 C144S n=4); Mann-Whitney test was performed to assess significance * p<0.05 compared to WT

Figure 4.. TRIM72 C144S does not impair whole body insulin sensitivity.

A. Glucose tolerance (WT n=12; TRIM72 C144S n=12) and B. insulin tolerance tests (WT n=10; TRIM72 C144S n=12) were performed on mice fasted for 6 hrs. Values are mean ± SEM; One-way ANOVA with repeated measures with Bonferroni posthoc test were performed to assess significance

Figure 5.. TRIM72 C144S mice do not have altered oxygen consumption and RER.

A. Oxygen consumption and B. RER were measured in mice with metabolic cages. Values are mean ± SEM; (WT n=5; TRIM72 C144S n=7); T-tests were performed to assess significance

Figure 6.. Expression of the E3 ubiquitin ligase targets of TRIM72 are not altered in TRIM72 C144S mouse hearts.

Protein expression of A. IRβ (WT n=3; TRIM72 C144S n=3), B. IRS1 (WT n=5; TRIM72 C144S n=5), C. FAK (WT n=3; TRIM72 C144S n=3) and D. HADH (WT n=5; TRIM72 C144S n=5) in WT and TRIM72 C144S mouse hearts. Values are mean ± SEM; T-tests were performed to assess significance

Figure 7.. Angiotensin II induced cardiac hypertrophy is not exacerbated in TRIM72 C144S mice.

A. Body weight (all groups n=6), B. heart weight (all groups n=6), C. heart weight/tibia length (all groups n=6), and D. ejection fraction (all groups n=5) of male WT mice treated with vehicle (WT+Veh) or Angiotensin II (WT+AngII) and male TRIM72 C144S mice treated with vehicle (TRIM72 C144S+Veh) or Angiotensin II (TRIM72 C144S+AngII). Two-way ANOVA with Bonferroni posthoc test were performed to assess significance, Values are mean ± SEM; * p<0.05 vs WT+Veh, ^> p<0.05 vs TRIM72 C144S+Veh