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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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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.
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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.

Mentions: Western blot results showed that type 2 and 3 IP3Rs were expressed in cardiomyocytes, while type 1 IP3R expression was undetectable (Fig. 1A). Similar to the results of the Western blot analysis, type 3 IP3R was distributed in the cytoplasm and intense perinuclear and intranuclear staining was evident for type 2 IP3R in immunofluorescence study, while type 1 IP3R was undetectable.


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)

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

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

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.
Mentions: Western blot results showed that type 2 and 3 IP3Rs were expressed in cardiomyocytes, while type 1 IP3R expression was undetectable (Fig. 1A). Similar to the results of the Western blot analysis, type 3 IP3R was distributed in the cytoplasm and intense perinuclear and intranuclear staining was evident for type 2 IP3R in immunofluorescence study, while type 1 IP3R was undetectable.

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