Calcium-sensing receptors regulate cardiomyocyte Ca2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation

Abstract

Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca2+ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca2+ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP3Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP3Rs were located in the SR. [Ca2+]i, [Ca2+]m and [Ca2+]SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca2+]SR, increased [Ca2+]i and [Ca2+]m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca2+ release from the SR into the mitochondria through IP3Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.

Figure 1

Subcellular IP₃Rs localization. (A) Immunocytochemical staining of cardiomyocyte with specific antibodies for type 1, type 2 and type 3 IP3Rs. (B) Western blot analysis of cardiomyocyte lysates using antibodies specific for IP3R, type 1, type 2 and type 3, respectively. DAPI and FITC to co-stain nuclei and type 3 IP₃ receptors and show the spatial relation between the two structures.

Figure 2

Viability of cardiomyocytes was examined using the MTT assay. The cell viability of the control was adjusted to 100%. The data presented are expressed as the mean ± SEM. *p < 0.05 vs Control group; †p < 0.05 vs Ca + Ni + Cd-H/Re .The experiment was repeated three times with similar results.

Figure 3

Hoechst-stained nuclei of apoptotic myocytes were analyzed morphologically and were expressed as the percentage of total nuclei. (magnification × 400). A: control group. B: H/Re group. C: Ca + Ni + Cd-H/Re group. D: NPS-2390 + Ca + Ni + Cd-H/Re. E: 2-APB + Ca + Ni + Cd-H/Re. F: Ru + Ca + Ni + Cd-H/Re group. G: Gd + Ca + Ni + Cd-H/Re The cardiomyocytes were placed in hypoxic culture medium for 3 h and then reoxygenated for 6 h by replacing hypoxic culture medium with fresh DMEM containing 10% FBS, and were treated with different inhibitors, respectively. The data presented are expressed as the mean ± SEM. *p < 0.05 vs Control group; †p < 0.05 vs Ca + Ni + Cd-H/Re.

Figure 4

The measurement of [Ca²⁺] after hypoxia/reoxygenation by laser confocal microscopy. (a) A: Control group. B: H/Re group. C: Ca + Ni + Cd-H/Re group. D: NPS-2390 + Ca + Ni + Cd-H/Re. E:2-APB + Ca + Ni + Cd-H/Re -H/Re. (b) Values represent the group mean ± SEM of at least four independent experiments. *p < 0.05 vs Control group; †p < 0.05 vs Ca + Ni + Cd-H/Re.

Figure 5

CaR activation induced Ca²⁺ release from the ER during H/Re. (A) a images represent the beginning of reperfusion (0 min). a' images represent 60 min after reperfusion. (B) Values represent the group mean ± SEM of at least four independent experiments. *p < 0.05 vs Control group; †p < 0.05 vs Ca + Ni + Cd-H/Re . White bar represents reoxygenation 0 min; grey bar represents reoxygenation 60 min.

Figure 6

The measurement of [Ca²⁺]m after 1 h of reoxygenation by laser confocal microscopy. A: control group. B: H/Re group. C: Ca + Ni + Cd-H/Re group. D: NPS-2390 + Ca + Ni + Cd-H/Re E: 2-APB + Ca + Ni + Cd-H/Re -H/Re. F: Ru + Ca + Ni + Cd-H/Re group. (B) Value represents the group mean ± SEM of at least four independent experiments. *p < 0.05 vs Control group; †p < 0.05 vs Ca + Ni + Cd-H/Re . (C) Effect of hypoxia/reoxygenation and CaR activation on △ψm in neonatal rat cardiomyocytes Summarized data for the relative changes of JC-1 fluorescence. Data are mean ± SEM. †p < 0.05 vs sham control group *p < 0.05 vs Ca + Ni + Cd-H/Re group.

Figure 7

The intact (A) and p20 (B) of BAP31 expression during H/Re. A: sham control group. B: H/Re group. C: Ca + Ni + Cd-H/Re group. D: NPS-2390 + Ca + Ni + Cd-H/Re. E: 2-APB + Ca + Ni + Cd-H/Re. The fold change values were mean ± SEM n = 3-4.*p < 0.05 vs control group †p < 0.05 vs H/Re (C)

Figure 8

Bax (A) and bak (B) translocation to the mitochondrial fractions in rat cardiomyocytes after H/Re. A: control group, B: H/Re group, C: Ca + Ni + Cd-H/Re group, D: NPS-2390 + Ca + Ni + Cd-H/Re group and E: Ru + Ca + Ni + Cd-H/Re group. The fold-change values are mean ± SEM, n = 3-4, *p < 0.05 vs. control group †p < 0.05 vs. H/Re (C). Black bar represented the fold change of bax; white bar represented the fold change of bak.

Figure 9

The release of cytochrome-C from mitochondrial fractions. A: control group. B: H/Re group. C: Ca + Ni + Cd-H/Re group. D: NPS-2390 + Ca + Ni + Cd-H/Re group. E: Ru + Ca + Ni + Cd-H/Re group. The fold change of cyt c values are mean ± SEM n = 3-4. *p < 0.05 vs control group †p < 0.05 vs H/Re.