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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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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.
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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.

Mentions: The p20-BAP31 protein has been shown to direct pro-apoptotic signals between the SR and the mitochondria, resulting in the insertion of bax and bak into the outer mitochondria membrane, homo-oligomerization and release of cyt c from the mitochondria [22]. Our results suggest that bax and bak translocation to the mitochondria was significantly increased in the H/Re (3.52 ± 0.31, 3.22 ± 0.28) and Ca + Ni + Cd-H/Re (3.16 ± 0.33, 3.44 ± 0.41) groups compared with the NPS-2390 + Ca + Ni + Cd-H/Re (1.86 ± 0.15, 1.77 ± 0.22) and Ru + Ca + Ni + Cd-H/Re (1.29 ± 0.17, 1.4 ± 0.18) groups (Fig. 8). Next, mitochondrial release of cytochrome c was analyzed to prove the role of the mitochondrial apoptotic pathway. It was found that cytochrome c from mitochondria in the H/Re (0.3 ± 0.05) and Ca + Ni + Cd-H/Re (0.25 ± 0.04) groups was significantly decreased compared with the control (1.0 ± 0.1), NPS-2390- + Ca + Ni + Cd-H/Re (0.75 ± 0.09) and Ru + Ca + Ni + Cd-H/Re (0.69 ± 0.08) groups (Fig. 9).


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)

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

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

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.
Mentions: The p20-BAP31 protein has been shown to direct pro-apoptotic signals between the SR and the mitochondria, resulting in the insertion of bax and bak into the outer mitochondria membrane, homo-oligomerization and release of cyt c from the mitochondria [22]. Our results suggest that bax and bak translocation to the mitochondria was significantly increased in the H/Re (3.52 ± 0.31, 3.22 ± 0.28) and Ca + Ni + Cd-H/Re (3.16 ± 0.33, 3.44 ± 0.41) groups compared with the NPS-2390 + Ca + Ni + Cd-H/Re (1.86 ± 0.15, 1.77 ± 0.22) and Ru + Ca + Ni + Cd-H/Re (1.29 ± 0.17, 1.4 ± 0.18) groups (Fig. 8). Next, mitochondrial release of cytochrome c was analyzed to prove the role of the mitochondrial apoptotic pathway. It was found that cytochrome c from mitochondria in the H/Re (0.3 ± 0.05) and Ca + Ni + Cd-H/Re (0.25 ± 0.04) groups was significantly decreased compared with the control (1.0 ± 0.1), NPS-2390- + Ca + Ni + Cd-H/Re (0.75 ± 0.09) and Ru + Ca + Ni + Cd-H/Re (0.69 ± 0.08) groups (Fig. 9).

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