Limits...
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.

Show MeSH

Related in: MedlinePlus

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

License
getmorefigures.php?uid=PMC2908572&req=5

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.

Mentions: We have demonstrated that CaCl2-activated CaR induces the increase of [Ca2+]i, but the origin of intracellular calcium remains unclear. We examined [Ca2+]SR by Fluo-5N staining. Fluo-5N is a low-affinity Ca2+ indicator (Kd = 400 μmol/L) that is only bright where [Ca2+] is very high, such as in the SR [15]. Rat neonatal cardiomyocytes were loaded with Fluo-5N and permeabilized with saponin. Irregularly distributed bright spots were seen in cardiomyocytes. The Fluo-5N signal was stable at the beginning of reperfusion (Fig. 5). At 60 min after reperfusion, the Fluo-5N signal was detected in the SR. We found that the fluorescence intensity in the SR in the Ca + Ni + Cd-H/Re (376 ± 44) and H/Re (399 ± 42) groups was significantly decreased compared to the control (648 ± 62), NPS-2390 + Ca + Ni + Cd-H/Re (562 ± 64) and 2-APB + Ca + Ni + Cd-H/Re (532 ± 51) groups. Luo et al. have previously demonstrated that 3 μM 2-APB inhibited IP3Rs and prevented PE-induced enhancement of Ca2+ sparks in neonatal cardiomyocytes [20]. Our study also suggests that 3 μM 2-APB may decrease [Ca2+]i through the inhibition of Ca2+ release from the SR via IP3R. Thus, 2-APB treatment could maintain the fluorescence intensity in the SR of cardiomyocytes during reperfusion. These results suggested that the activation of CaR by CaCl2 or H/Re induced SR release of Ca2+.


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)

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

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

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.
Mentions: We have demonstrated that CaCl2-activated CaR induces the increase of [Ca2+]i, but the origin of intracellular calcium remains unclear. We examined [Ca2+]SR by Fluo-5N staining. Fluo-5N is a low-affinity Ca2+ indicator (Kd = 400 μmol/L) that is only bright where [Ca2+] is very high, such as in the SR [15]. Rat neonatal cardiomyocytes were loaded with Fluo-5N and permeabilized with saponin. Irregularly distributed bright spots were seen in cardiomyocytes. The Fluo-5N signal was stable at the beginning of reperfusion (Fig. 5). At 60 min after reperfusion, the Fluo-5N signal was detected in the SR. We found that the fluorescence intensity in the SR in the Ca + Ni + Cd-H/Re (376 ± 44) and H/Re (399 ± 42) groups was significantly decreased compared to the control (648 ± 62), NPS-2390 + Ca + Ni + Cd-H/Re (562 ± 64) and 2-APB + Ca + Ni + Cd-H/Re (532 ± 51) groups. Luo et al. have previously demonstrated that 3 μM 2-APB inhibited IP3Rs and prevented PE-induced enhancement of Ca2+ sparks in neonatal cardiomyocytes [20]. Our study also suggests that 3 μM 2-APB may decrease [Ca2+]i through the inhibition of Ca2+ release from the SR via IP3R. Thus, 2-APB treatment could maintain the fluorescence intensity in the SR of cardiomyocytes during reperfusion. These results suggested that the activation of CaR by CaCl2 or H/Re induced SR release of Ca2+.

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