Catecholamine Surges Cause Cardiomyocyte Necroptosis via a RIPK1–RIPK3-Dependent Pathway in Mice

Figure 1. Isoproterenol (ISO) treatment induced cardiomyocyte necrosis in wild-type mice. (A) A schematic for the design of experiments presented in this figure and in Figures 2, 3. Adult mice of both sexes were subjected to two daily subcutaneous injections of ISO (85 mg/kg) or vehicle control [saline (SAL)] with an interval of 24 h. Myocardial tissue was collected at 24 h after the second dose of ISO. Evans blue dye (EBD) was injected 18 h before tissue collection. EBD in the vasculature and extracellular space was flushed out through retrograde perfusion with saline via the abdominal aorta immediately before tissue collection. A separate cohort without EBD injection and perfusion fixation was used for biochemical analyses. (B,C) in vivo EBD uptake assays. Cryosections were used for staining with Alexa 488-conjugated phalloidin and DAPI before imaging with a multicolor confocal microscope. Shown are confocal micrographs centered on an EBD positive area in the ISO-treated heart or the corresponding region of a saline-treated heart (B) and a graph summarizing the percentage EBD-positive area from three mice (two males and one female) per group (C). Scale bar = 200 μm; mean ± SEM; three…

Figure 2. Myocardial RIPK1–RIPK3–MLKL pathway is activated in isoproterenol (ISO)-treated mice. Mice were treated with ISO or saline (SAL) as described in Figure 1. Ventricular myocardial samples were collected at 24 h after the second dose of ISO for further analyses, and total proteins were extracted for Western blotting analyses for the indicated proteins. (A–G) Representative images (A) and pooled densitometry data (B–G) of Western blotting analysis for the indicated proteins. p-S166-RIPK1, Ser166-phosphorylated RIPK1; p-S321-RIPK1, Ser321-phosphorylated RIPK1; p-MLKL, Ser345-phosphorylated MLKL; t-MLKL, total MLKL; L.C., loading control that used the stain-free total protein image; mean ± SEM; each dot represents a mouse; n = 3 mice (two males and one female) per group; p-values shown in this figure are derived from two-sided unpaired t-tests with Welch's correction. (H) Western blotting analysis (IB) for RIPK1 and RIPK3 that were immunoprecipitated (IP) with IgG or anti-RIPK1 antibodies.

Figure 3. RIPK3 knockout significantly reduced isoproterenol (ISO)-induced cardiomyocyte necrosis. Wild-type and RIPK3 null (RIPK3−/−) mice were subjected to the same ISO or saline (SAL) treatment regime and the Evans blue dye (EBD) uptake assays as described in Figure 1. (A,B) Representative confocal micrographs (A) and pooled morphometric data (B) of EBD-positive area. Scale bar = 200 μm. For the EBD assays, three mice (one male and two females) per group and three representative sections per heart were used for morphometry. The p-value is shown above the bracket and was obtained from nested t-test to account for the three technical repeats within each biological repeat. (C–E) Western blotting analyses for myocardial levels of the indicated proteins. Shown are representative image (C) and pooled densitometry data (D,E). L.C., loading control that used the stain-free total protein image; mean ± SEM; n = 4 mice (two males and two females) per group; two-way ANOVA followed by Tukey's test.

Figure 4. RIPK1 kinase activity is required for the isoproterenol (ISO) treatment to increase RIPK3 and p-MLKL in mouse hearts. (A) A schematic for the design of experiments presented in Figures 4, 5. Adult mice were subject to ISO or saline (SAL) treatment as illustrated. Necrostatin-1 (NEC-1; 4 mg/kg, i.p.) or vehicle control (DMSO) was administered 10min before each ISO or SAL injection. (B–G) Western blotting analyses for myocardial levels of the indicated proteins. Ventricular myocardium of wild-type (WT) mice was collected 24 h after the second ISO injection. Shown are representative images (B) and pooled quantitative data for the indicated proteins (C–G). Two-way ANOVA followed by Tukey's test was used; mean ± SEM; n = 3 mice (either two males + one female or one male + two females) per group. L.C. (loading control) used total protein images obtained with the stain-free total protein imaging technology.

Figure 5. Effect of necrostatin-1 (NEC-1) on isoproterenol (ISO)-induced cardiomyocyte necrosis in wild-type (WT) and RIPK3 knockout mice. WT and RIPK3 null mice were subjected to NEC-1 and ISO treatments as well as the EBD injection as described in Figure 4A. Perfusion-fixed myocardial samples were collected 24 h after the second ISO injection for EBD uptake assays. Shown are representative images (A; scale bar = 500 μm) and pooled quantitative data (B) from the EBD uptake assays. Two representative sections per mouse and three mice (two males and one female) per group were analyzed; mean ± SEM, nested ANOVA followed by Tukey's test. The nested test is used to account for the two technical repeats (two sections per mouse).

Figure 6. A working model for the potential mechanisms by which high doses of isoproterenol (ISO) induce cardiomyocyte necroptosis. The pathological processes are illustrated to occur sequentially in two adjacent cardiomyocytes (A and B), but they could potentially take place in the same cell where TNFα and other inflammatory cytokines would be autocrinal. Processes shown in cell A are based on previous reports. The pathway modeled in cell B is based on the findings of the present study, but the involvement of TNFα and its receptor (shaded area at the top) as well as the requirement of MLKL for the induction of cardiomyocyte necrosis by ISO remain to be fully established. I/R, ischemia/reperfusion; TNFR1, TNFα receptor type 1; RIPK3-KO, RIPK3 knockout; NEC-1, necrostatin-1.