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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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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.
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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.

Mentions: To confirm the role of CaR in cardiomyocyte apoptosis evoked by H/Re, we examined whether activation of CaR induced apoptosis in cultured cardiomyocytes of neonatal rats under our experimental conditions. We used two CaR agonists, CaCl2 and GdCl3, to demonstrate the role of CaR in the induction of apoptosis during H/Re. When cardiomyocytes were exposed to the activation of CaR by H/Re, cell viability was shown to be reduced to 80.2 ± 4.8% (H/Re), 78.3 ± 6.8% (Ca + Ni + Cd-H/Re) and 77.6 ± 5.1% (Gd + Ni + Cd-H/Re), respectively, compared with that of control cells using the MTT assay. Cell viability in NPS-2390 + Ca + Ni + Cd-H/Re (91.7 ± 4.6%), NPS-2390 is an allosteric antagonist of group 1 metabotropic glutamate receptors. 2-APB + Ca + Ni + Cd-H/Re (88.3 ± 5.2%, 2-APB is a selective inhibitor) and Ru + Ca + Ni + Cd-H/Re (87.6 ± 5.6%, Ruthenium red is an inhibitor of mitochondrial calcium uniporter) groups was more than that of the H/Re, Ca + Ni + Cd-H/Re and Gd + Ni + Cd-H/Re groups (Fig. 2).


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)

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

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

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.
Mentions: To confirm the role of CaR in cardiomyocyte apoptosis evoked by H/Re, we examined whether activation of CaR induced apoptosis in cultured cardiomyocytes of neonatal rats under our experimental conditions. We used two CaR agonists, CaCl2 and GdCl3, to demonstrate the role of CaR in the induction of apoptosis during H/Re. When cardiomyocytes were exposed to the activation of CaR by H/Re, cell viability was shown to be reduced to 80.2 ± 4.8% (H/Re), 78.3 ± 6.8% (Ca + Ni + Cd-H/Re) and 77.6 ± 5.1% (Gd + Ni + Cd-H/Re), respectively, compared with that of control cells using the MTT assay. Cell viability in NPS-2390 + Ca + Ni + Cd-H/Re (91.7 ± 4.6%), NPS-2390 is an allosteric antagonist of group 1 metabotropic glutamate receptors. 2-APB + Ca + Ni + Cd-H/Re (88.3 ± 5.2%, 2-APB is a selective inhibitor) and Ru + Ca + Ni + Cd-H/Re (87.6 ± 5.6%, Ruthenium red is an inhibitor of mitochondrial calcium uniporter) groups was more than that of the H/Re, Ca + Ni + Cd-H/Re and Gd + Ni + Cd-H/Re groups (Fig. 2).

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