CaM promotes cardiomyocyte mitophagy in myocardial ischemia-reperfusion injury involving in the regulation of the IP3R3-GRP75-VDAC1 complex

Figures

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  Fig. 1. MIRI induces upregulation of the IP3R3-GRP75-VDAC1 complex proteins and mitochondrial Ca2+ overload in cardiomyocytes. (A). IP3R3, GRP75 and VDAC1 protein expression; (B, C and D). The relative ratio; (E).Rhod-2 fluorescence; (F).Mitochondrial calcium content; (G) ATP level; (H). Mitochondrial membrane potential; (I) Fold-change. A post-hoc test was used following the one-way ANOVA.*P < 0.05 vs. sham; **P < 0.01 vs. sham. IP3R3 receptor for inositol 1,4,5-trisphosphate, GRP75 glucose-regulated protein 75,, VDAC1 voltage-dependent anion-selective channel 1, ANOVA analysis of variance.

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  Fig. 2. MIRI induces mitophagy and cell injury in cardiomyocytes. (A). CaM mRNA expression levels; (B). CaM, ATG4, LC3-2, and LC3-1 protein expression; (C, D, and E). The corresponding quantification analysis of protein expression; (F). CK activity; (G) TnT levels; (H). LDH levels; (I). TUNEL/Hoechst double staining. (J). TUNEL positive cell counting. (K). Co-localization of mitochondria and LC3. A post-hoc test was used following the one-way ANOVA analysis. *P < 0.05 vs. sham;**P < 0.01 vs. sham;***P < 0.001 vs. sham. MIRI myocardial ischemia-reperfusion injury, CaM calmodulin, ATG4 autophagy-regulating protease 4, LC3 microtubule-associated protein 1 A/1B-light chain 3, CK creatine kinase, TnT troponin-T, LDH lactate dehydrogenase, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, ANOVA analysis of variance.

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  Fig. 3. GRP75 knockdown attenuates H/R-induced mitochondrial calcium overload in H9C2 cells. (A) and (B). GRP75 mRNA expression levels; (C) IP3R3, GRP75, and VDAC1 protein expression; (D, E, and F). The corresponding quantification analysis of protein expression; (G). Rhod-2 fluoresence; (H).Mitochondrial calcium content; (I). ATP levels; (J). Mitochondrial membrane potential; (K). Fold-change. A post-hoc test was used following the one-way ANOVA analysis. **P < 0.01 vs. control; ***P < 0.01 vs. NC siRNA;##P < 0.01 vs. H/R; ###P < 0.001 vs. H/R. GRP75 glucose-regulated protein 75, H/R hypoxia/reoxygenation, IP3R3 receptor for inositol 1,4,5-trisphosphate, VDAC1 voltage-dependent anion-selective channel 1, ANOVA analysis of variance.

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  Fig. 4. GRP75 knockdown attenuates H/R-induced mitophagy in H9C2 cells. (A). CaM, ATG4, LC3-2, and LC3-1 protein expression; (B). The corresponding quantification analysis of CaM protein expression; (C) ATG4 mRNA expression; (D). The corresponding quantification analysis of ATG4 protein expression; (E) ATG4 mRNA expression; (F). The corresponding quantification analysis of the ratio of LC3-2 to LC3-1; (G). LDH levels; (H). TUNEL/Hoechst double staining. (I). TUNEL positive cell counting. (J). Co-localization of mitochondria and LC3. Post-hoc test was used following the one-way ANOVA analysis. *P < 0.05 vs. control;**P < 0.01 vs. control; #P < 0.05 vs. H/R. GRP75 glucose-regulated protein 75, H/R hypoxia/reoxygenation, CaM calmodulin, ATG4 autophagy-regulating protease 4, LC3 microtubule-associated protein 1 A/1B-light chain 3, LDH lactate dehydrogenase, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, ANOVA analysis of variance.

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  Fig. 5. CaM knockdown exhibits no apparent effect on H/R-induced mitochondrial calcium overload in H9C2 cells. (A) and (B). CaM mRNA expression levels; (C). CaM, IP3R3, GRP75, and VDAC1 protein expression levels; (D, E, F, and G). The corresponding quantification analysis of protein expression; (H). Rhod-2 fluoresence; (I). Mitochondrial calcium content; (J). ATP levels; (K): mitochondrial membrane potential; (L). fold-change. A post-hoc test was used following the one-way ANOVA analysis.*P < 0.05 vs. control; **P < 0.01 vs. control; ***P < 0.01 vs. NC siRNA;##P < 0.01 vs. H/R; ###P < 0.001 vs. H/R. CaM calmodulin,H/R hypoxia/reoxygenation, IP3R3 receptor for inositol 1,4,5-trisphosphate, GRP75 glucose-regulated protein 75, VDAC1 voltage-dependent anion-selective channel 1, ANOVA analysis of variance.

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  Fig. 6. Effects of CaM knockdown on H/R-induced mitophagy in H9C2 cells. (A). ATG4, LC3-2, and LC3-1 protein expression; (B and C). The corresponding quantification analysis of protein expression; (D). LDH levels; (E). TUNEL/Hoechst double staining; (F). TUNEL positive cell counting. (G). Co-localization of mitochondria and LC3. A post-hoc test was used following the one-way ANOVA test.*P < 0.05 vs. control; **P < 0.01 vs. control; #P < 0.05 vs. H/R. ##P < 0.01 vs. H/R. CaM, calmodulin,H/R, hypoxia/reoxygenation, ATG4, autophagy-regulating protease 4,LC3, microtubule-associated protein 1 A/1B-light chain 3, LDH, lactate dehydrogenase, TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling, ANOVA, analysis of variance.

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  Fig. 7. Schematic diagram of MIRI. MIRI led to an upregulated expression of the IP3R3-GRP75-VDAC1 complex proteins in cardiomyocytes, which resulted in mitochondrial calcium overload. In addition, MIRI led to an upregulated in the expression levels of CaM. The Ca2+ bind to CaM forming the Ca2+-CaM complex, which induces mitophagy-related gene (LC3 and ATG4) expression. This in turn leads to induction of mitophagy. Excessive mitophagy results in cell damage. MIRI myocardial ischemia-reperfusion injury, IP3R3 receptor for inositol 1,4,5-trisphosphate, GRP75 glucose-regulated protein 75, VDAC1 voltage-dependent anion-selective channel 1, CaM calmodulin; Ca2+, calcium ions, LC3 microtubule-associated protein 1 A/1B-light chain 3, ATG4 autophagy-regulating protease 4.