MLKL–Mediated Necroptosis Predominantly Contributes to Immune-Associated Myocardial Damage

Figures

• 

  Fig. 1. Characterization of immune activation-induced cardiomyocyte injury a The impedance of hiPSC-CMs treated with aPBMCs. Mean ± SD in the scatter plot, n = 6 b The EFP waveforms of hiPSC-CMs captured at pretreatment and 3 min and 6, 12, 24 and 48 h, and wash out after treatment with increasing E:T ratios of aPBMCs. c LDH release in the supernatant of hiPSC-CMs was determined using luciferase assays after aPBMCs treated for 24 and 48 h. Mean ± SD in the scatter plot, n = 3 d Morphology and viability of GFP-CMs (green) were monitored using IncuCyte live cell fluorescence imaging system during co-culture with aPBMCs. Scale bar: 400 μm (red) or 125 μm (yellow). Mean ± SD in the scatter plot, n = 3 e Western blot analysis of PD-L1 in GFP-CMs after treatment with aPBMCs at an E:T ratio of 5:1 for 48 h. Mean ± SD, n = 3 *P < 0.5, **P < 0.01, ***P < 0.001 vs CON

• 

  Fig. 2. Death pathways in cardiomyocyte death induced by immune activation a–c Transcriptome analysis of GFP-CMs with or without treatment with aPBMCs for 6 h. a BP analysis of GO terms in transcriptomic data. b KEGG enrichment analysis of the top 20 pathways and 4 death pathways. c GSEA of death pathways based on KEGG enrichment analysis. n = 3 in each group. d Dynamic immunofluorescence staining of key proteins in different death pathways of GFP-CMs. GFP-CMs with or without treatment with aPBMCs for 6, 12, and 24 h were fixed for immunofluorescence staining with key proteins of pyroptosis, apoptosis, and necroptosis. NLRP3 (pyroptosis), GSDMD (pyroptosis), cleaved caspase- 3 (apoptosis), and MLKL (necroptosis) are stained in red with an AF594-conjugated secondary antibody. Nuclei are stained with Hoechst (blue). CMs are marked with GFP (green). Scale bar: 100 μm (white). Mean ± SD. For NLRP3 analysis, n = 84 in the CON group, n = 76, 78, and 74 in the Co-aPBMC groups of co-cultured with aPBMC for 6, 12 and 24 h, respectively. For GSDMD analysis, n = 75 in the CON group, n = 82, 78, and 72 in the Co-aPBMC groups of co-cultured with aPBMC for 6, 12 and 24 h, respectively. For cleaved…

• 

  Fig. 3. Inhibition of necroptosis rescued cardiomyocytes from immune injury a Schematic diagram of pharmacological blockade strategies and their targets. Key proteins in pyroptosis, apoptosis, necroptosis, and ferroptosis are represented by orange, purple, green, and red ellipses, respectively. Inhibitors are represented by blue rectangles. HiPSC-CMs (or GFP-CMs) were pretreated with MCC (pyroptosis inhibitor, targeting NLRP3, 10 μM), Emr (apoptosis inhibitor, targeting caspase, 10 μM), NSA (necroptosis inhibitor, targeting MLKL, 10 μM), Nec- 1 (necroptosis inhibitor, targeting RIPK1, 10 μM) and GSK872 (necroptosis inhibitor, targeting RIPK3, 10 μM) for 6 h, respectively, followed by co-culturing with aPBMCs for 48 h. b The viability (impedance) in hiPSC-CMs. Mean ± SD, n = 4 c LDH release in the supernatant of hiPSC-CMs. Mean ± SD, n = 3 d GFP-CMs (green) morphology and viability were monitored using the IncuCyte live cell fluorescence imaging system during co-culture with aPBMCs. Scale bar: 400 μm (red) or 125 μm (yellow). Mean ± SD, n = 4 *P < 0.05, **P < 0.01, ***P < 0.001 vs CON.##P < 0.01, ###P < 0.001 vs vehicle

• 

  Fig. 4. Knockdown of MLKL rescued cardiomyocytes from immune injury a The MLKL knockdown efficiency of hiPSC-CMs was determined by qRT–PCR. Mean ± SD, n = 3 b The MLKL knockdown efficiency of hiPSC-CMs was determined by Western blot. Mean ± SD, n = 3 c The impedance of hiPSC-CMs with MLKL knockdown co-cultured with aPBMCs for 48 h. Mean ± SD in the scatter plot, n = 3 d LDH release in the supernatant of MLKL knockdown hiPSC-CMs was determined using luciferase assay after treatment with aPBMCs for 48 h. Mean ± SD, n = 4 e The morphology and viability of MLKL knockdown GFP-CMs (green) were monitored using the IncuCyte live cell fluorescence imaging system during co-culture with aPBMCs. Scale bar: 400 μm (red) or 125 μm (yellow). Mean ± SD, n = 3 **P < 0.01, ***P < 0.001 vs CON.#P < 0.05, ##P < 0.01,###P < 0.001 vs NC

• 

  Fig. 5. Characterization of ICIs-induced cardiac immune injury in mice a Serum markers of myocardial injury (cTnI and CKMB) in mice treated with IgG or ICIs for 30 days. Mean ± SD, n = 11 in each group. b Western blot analysis of PD-L1 and IL- 6 in heart tissue. Mean ± SD, n = 5 in IgG, n = 6 in ICIs. c Summary data regarding the incidence of myocardial necrosis. n = 11 d Representative images of H&E staining and Masson’s staining of heart tissues. Scale bar: 1 mm (black) or 50 μm (blue). e Representative images of immunohistochemical staining using T cell surface molecules, including CD4, CD8, and CD44. Red, blue, and green arrows indicate CD4+, CD8+, and CD44+ cells, respectively. Scale bar: 1 mm (black) or 40 μm (red). f Representative images of immunofluorescence staining of markers of pyroptosis, apoptosis, and necroptosis in mouse heart tissues treated with ICIs. NLRP3 (pyroptosis), GSDMD (pyroptosis), cleaved caspase- 3 (apoptosis), and MLKL (necroptosis) are stained in red. Nuclei are stained with DAPI (blue). Cell membranes are marked with WGA (green). Scale bar: 1 mm or 200 μm. Mean ± SD, n = 3 *P < 0.05, **P < 0.01, ***P < 0.001 vs IgG

• 

  Fig. 6. Inhibition of MLKL prevented ICIs-induced cardiac immune injury in mice a Schematic diagram of GW80 treatment. b Serum markers of myocardial injury (cTnI and CKMB) in mice, n = 10 c Summary data regarding the incidence of myocardial necrosis. d Representative images of H&E staining and Masson’s staining of heart tissues. Scale bar: 500 μm (black) or 100 μm (blue). e Representative images of immunofluorescence staining of proteins involved in pyroptosis, apoptosis, and necroptosis in mouse right ventricular wall. NLRP3 (pyroptosis), GSDMD (pyroptosis), cleaved caspase- 3 (apoptosis), and MLKL (necroptosis) are stained in red. Nuclei are stained with DAPI (blue). Cell membranes are marked with WGA (green). Scale bar: 100 μm (white). Mean ± SD, n = 3 *P < 0.05, **P < 0.01, ***P < 0.001 vs IgG. #P < 0.05, ##P < 0.01, ###P < 0.001 vs ICIs + vehicle.&P < 0.05 vs IgG + GW80