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

Lu FH, Tian Z, Zhang WH, Zhao YJ, Li HL, Ren H, Zheng HS, Liu C, Hu GX, Tian Y, Yang BF, Wang R, Xu CQ - J. Biomed. Sci. (2010)

Bottom Line: 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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathophysiology, Harbin Medical University, Harbin 150086, China.

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.

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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.
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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.

Mentions: To further determine whether the cell death induced by H/Re and activation of CaR was mediated by apoptosis, the nuclear morphology was analyzed using the Hoechst staining assay. The apoptotic cells exhibited typical fragmented nuclei and condensed chromatin on staining with Hoechst 33342 (Fig. 3). The percentage of apoptotic cells relative to the total number of cells was increased to H/Re (33 ± 6%), Ca + Ni + Cd-H/Re (31 ± 5%) and Gd + Ni + Cd-H/Re (34 ± 3%) compared with the NPS-2390 + Ca + Ni + Cd-H/Re (20 ± 4%), 2-APB + Ca + Ni + Cd-H/Re (18 ± 4%) and Ru + Ca + Ni + Cd-H/Re (23 ± 5%) groups. Therefore, these data show that the activation of CaR is involved in H/Re - induced cardiomyocyte apoptosis.


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

Lu FH, Tian Z, Zhang WH, Zhao YJ, Li HL, Ren H, Zheng HS, Liu C, Hu GX, Tian Y, Yang BF, Wang R, Xu CQ - J. Biomed. Sci. (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2908572&req=5

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.
Mentions: To further determine whether the cell death induced by H/Re and activation of CaR was mediated by apoptosis, the nuclear morphology was analyzed using the Hoechst staining assay. The apoptotic cells exhibited typical fragmented nuclei and condensed chromatin on staining with Hoechst 33342 (Fig. 3). The percentage of apoptotic cells relative to the total number of cells was increased to H/Re (33 ± 6%), Ca + Ni + Cd-H/Re (31 ± 5%) and Gd + Ni + Cd-H/Re (34 ± 3%) compared with the NPS-2390 + Ca + Ni + Cd-H/Re (20 ± 4%), 2-APB + Ca + Ni + Cd-H/Re (18 ± 4%) and Ru + Ca + Ni + Cd-H/Re (23 ± 5%) groups. Therefore, these data show that the activation of CaR is involved in H/Re - induced cardiomyocyte apoptosis.

Bottom Line: 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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathophysiology, Harbin Medical University, Harbin 150086, China.

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.

Show MeSH
Related in: MedlinePlus