Fig 1: Binding of ERP44 to IP3R1 inhibits the I/R-induced mitochondrial Ca2+ overload.A, C Western blot was used to detect the level of ERP44 protein; B immunoprecipitation was used to detect the binding relationship between IP3R1 and ERP44; D Rhod2 AM fluorescent labeling was used to observe the changes of intracellular Ca2+ level. Compared to the blank group, *p < 0.05, ***p < 0.001; for pairwise comparison, ##p < 0.01, ###p < 0.001. All experiments were performed three times independently. Data were analyzed using one-way ANOVA and Tukey’s multiple comparison test.
Fig 2: Inhibition of IP3R1 reduces MI/R-induced pyroptosis and alleviates myocardial injury.A RT-qPCR and western blot were used to detect the levels of IP3R1 in the myocardium of rats after MI/R and shRNA intervention; B TTC staining was used to detect the infarct size of MI/R rats. AAR/LV reflects the degree of ischemia, INF/AAR reflects the degree of infarction, n = 3; C HE staining was used to observe the pathological changes of myocardium of MI/R rats, n = 6. D the effect of IP3R1 on the concentration of myocardial enzymes in MI/R rats, n = 21; E immunohistochemical observation of GSDMD-positive expression, n = 6; F colorimetric method was used to detect the activity of Caspase-1, n = 6; G Western blot detected the levels of NLRP3, ASC, and GSDMD-N, n = 6; H ELISA was used to measure the levels of IL-1ß and IL-18, n = 6; compared with the sham group, ***p < 0.001; for pairwise comparison, ###p < 0.001. Data in panels A/B/D/F were analyzed using one-way ANOVA and data in panels G/H were analyzed using two-way ANOVA. Tukey’s multiple comparison test was applied for the post hoc test after ANOVA.
Fig 3: Inhibition of IP3R1 attenuates H/R-induced pyroptosis and inflammation in cardiomyocytes.A RT-qPCR was used to detect the efficiency of siRNA transfection; B CCK-8 was used to detect cell viability; C Western blot was used to detect the levels of pyroptosis-related proteins; D colorimetric method was used to detect the activity of Caspase-1; E ELISA was used to detect the levels of IL-1ß and IL-18. Cardiomyocytes are isolated and cultured from adult rat myocardium. Compared to the blank group, *p < 0.05, **p < 0.01, ***p < 0.001; for pairwise comparison, #p < 0.05, ##p < 0.01, ###p < 0.001. Data in panels A/D were analyzed using one-way ANOVA and data in panels B/C/E were analyzed using two-way ANOVA. Tukey’s multiple comparison test was applied for the post hoc test after ANOVA.
Fig 4: Upregulation of IP3R1 leads to intracellular Ca2+ overload in cardiomyocytes.A, B Fluo 4-AM and Rhod2 AM were used to observe the changes of Ca2+ levels in cells and mitochondria; n = 6. Compared with the sham group, *p < 0.05, **p < 0.01, ***p < 0.001; for pairwise comparison, #p < 0.05, ##p < 0.01, ###p < 0.001. Data were analyzed using one-way ANOVA and Tukey’s multiple comparison test.
Fig 5: The pleiotropic roles of TRPCs in regulating CaM and IP3R1.a The Co-IP analysis of TRPC1 or TRPC6 binding with IP3R1 in mice heart tissues (pooled tissues from 3 male mice per sample, n = 2 biological independent experiments). b The interaction between CaM and IP3R1 in the hearts of WT, Trpc1−/−, and Trpc6−/− mice 4 h after LPS challenge (pooled tissues from 3 male mice per sample, n = 2 biological independent experiments). c TRPC1-IP3R1 and TRPC6-IP3R1 interactions in LPS-challenged neonatal WT mice cardiomyocytes. Representative PLA photomicrographs (left panel) and the statistical analysis (right panel) are shown (mean ± SEM, n = 6 images from 3 biological independent experiments). Statistical significance was determined using the two-tailed Student’s t-test. Exact P value = 2.0 × 10−5 (Trpc1−/− vs Trpc1−/−+LPS) and 3.5 × 10−5 (Trpc6−/− vs Trpc6−/−+LPS). d IP3R1-CaM interactions in LPS-challenged neonatal WT, Trpc1−/−, and Trpc6−/− mice cardiomyocytes. Representative PLA photomicrographs (upper panel) and the statistical analysis (lower panel) are shown (mean ± SEM, n = 6 images from 3 biological independent experiments). Statistical significance was determined using the one-way ANOVA with Tukey’s multiple comparisons test. Exact P value = 8.3 × 10−13 (WT + LPS vs Trpc1−/−+LPS) and 9.6 × 10−13 (WT + LPS vs Trpc6−/−+LPS). e Representative immunofluorescent photomicrographs of TRPC1, CaM, IP3R1, and PDI (upper panel) and traces of fluorescence intensity spatial profiles of IP3R1, TRPC1, and PDI (lower panel) in LPS-stimulated neonatal mice cardiomyocytes (n = 6 images from 3 biological independent experiments). f The effects of W-7 on the LPS-triggered intracellular Ca2+ influx in adult mice cardiomyocytes (left panel) and mice bone marrow-derived macrophages (right panel) are shown (mean ± SEM, n = 15–20 cells from 3 male mice per group). Statistical significance was determined using the one-way ANOVA with Game Howell’s multiple comparisons test. In cardiomyocytes, exact P value = 8.4 × 10−10 (Trpc1−/− vs Trpc1−/−+W-7) and 1.3 × 10−8 (Trpc6−/− vs Trpc6−/−+W-7. Source data are provided as a Source Data file.
Supplier Page from Abcam for Anti-IP3R1 antibody