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Impact of hypoxia, simulated ischemia and reperfusion in HL-1 cells on the expression of FKBP12/FKBP12.6 and intracellular calcium dynamics

Astrom-Olsson, Karin; Li, Lizhen; Olofsson, Charlotta S.; Boren, Jan; Öhlin, Hans LU and Grip, Lars (2012) In Biochemical and Biophysical Research Communications 422(4). p.732-738
Abstract
Aims: To establish a cardiac cell culture model for simulated ischemia and reperfusion and in this model investigate the impact of simulated ischemia and reperfusion on expression of the calcium handling proteins FKBP12 and FKBP12.6, and intracellular calcium dynamics. Methods: HL-1 cell cultures were exposed to normoxia (as control), hypoxia, simulated ischemia (HEDA) or HEDA + reactive oxygen species (ROS) for up to 24 h and after HEDA, with or without ROS, followed or not by simulated reperfusion (REPH) for 6 h. Viability was analyzed with a trypan blue exclusion method. Cell lysates were analyzed with real-time PCR and Western blot (WB) for FKBP12 and FKBP12.6. Intracellular Ca(2+)measurements were performed using dual-wavelength ratio... (More)
Aims: To establish a cardiac cell culture model for simulated ischemia and reperfusion and in this model investigate the impact of simulated ischemia and reperfusion on expression of the calcium handling proteins FKBP12 and FKBP12.6, and intracellular calcium dynamics. Methods: HL-1 cell cultures were exposed to normoxia (as control), hypoxia, simulated ischemia (HEDA) or HEDA + reactive oxygen species (ROS) for up to 24 h and after HEDA, with or without ROS, followed or not by simulated reperfusion (REPH) for 6 h. Viability was analyzed with a trypan blue exclusion method. Cell lysates were analyzed with real-time PCR and Western blot (WB) for FKBP12 and FKBP12.6. Intracellular Ca(2+)measurements were performed using dual-wavelength ratio imaging in fura-2 loaded cells. Results: A time-dependent drop in viability was shown after HEDA (P < 0.001). Viability was not further influenced by addition of ROS or REPH. The general patterns of FKBP12 and FKBP12.6 mRNA expression showed upregulation after hypoxia, downregulation after ischemia and normalization after reperfusion, which was partially attenuated if ROS was added during HEDA. The protein contents were unaffected after hypoxia, tended to increase after ischemia and, for FKBP12.6, a further increase after reperfusion was shown. Hypoxia or HEDA, with or without REPH, resulted in a decreased amplitude of the Ca2+ peak in response to caffeine. In addition, cells subjected to HEDA for 3 h or HEDA for 3 h followed by 6 h of REPH displayed irregular Ca2+ oscillations with a decreased frequency. Conclusion: A threshold for cell survival with respect to duration of ischemia was established in our cell line model. Furthermore, we could demonstrate disturbances of calcium handling in the sarcoplasmic reticulum as well as alterations in the expressions of the calcium handling proteins FKBP12 and FKBP12.6, why this model may be suitable for further studies on ischemia and reperfusion with respect to calcium handling of the sarcoplasmic reticulum. (C) 2012 Elsevier Inc. All rights reserved. (Less)
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author
organization
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Contribution to journal
publication status
published
subject
keywords
HL-1 cells, FKBP12/FKBP12.6, Viability, Ca2+
in
Biochemical and Biophysical Research Communications
volume
422
issue
4
pages
732 - 738
publisher
Elsevier
external identifiers
  • wos:000306443900036
  • scopus:84862296457
ISSN
1090-2104
DOI
10.1016/j.bbrc.2012.05.071
language
English
LU publication?
yes
id
8dafe6f1-eb3a-411f-86bb-d6ff210e5bea (old id 2991719)
date added to LUP
2012-09-03 07:17:23
date last changed
2017-09-17 06:19:53
@article{8dafe6f1-eb3a-411f-86bb-d6ff210e5bea,
  abstract     = {Aims: To establish a cardiac cell culture model for simulated ischemia and reperfusion and in this model investigate the impact of simulated ischemia and reperfusion on expression of the calcium handling proteins FKBP12 and FKBP12.6, and intracellular calcium dynamics. Methods: HL-1 cell cultures were exposed to normoxia (as control), hypoxia, simulated ischemia (HEDA) or HEDA + reactive oxygen species (ROS) for up to 24 h and after HEDA, with or without ROS, followed or not by simulated reperfusion (REPH) for 6 h. Viability was analyzed with a trypan blue exclusion method. Cell lysates were analyzed with real-time PCR and Western blot (WB) for FKBP12 and FKBP12.6. Intracellular Ca(2+)measurements were performed using dual-wavelength ratio imaging in fura-2 loaded cells. Results: A time-dependent drop in viability was shown after HEDA (P &lt; 0.001). Viability was not further influenced by addition of ROS or REPH. The general patterns of FKBP12 and FKBP12.6 mRNA expression showed upregulation after hypoxia, downregulation after ischemia and normalization after reperfusion, which was partially attenuated if ROS was added during HEDA. The protein contents were unaffected after hypoxia, tended to increase after ischemia and, for FKBP12.6, a further increase after reperfusion was shown. Hypoxia or HEDA, with or without REPH, resulted in a decreased amplitude of the Ca2+ peak in response to caffeine. In addition, cells subjected to HEDA for 3 h or HEDA for 3 h followed by 6 h of REPH displayed irregular Ca2+ oscillations with a decreased frequency. Conclusion: A threshold for cell survival with respect to duration of ischemia was established in our cell line model. Furthermore, we could demonstrate disturbances of calcium handling in the sarcoplasmic reticulum as well as alterations in the expressions of the calcium handling proteins FKBP12 and FKBP12.6, why this model may be suitable for further studies on ischemia and reperfusion with respect to calcium handling of the sarcoplasmic reticulum. (C) 2012 Elsevier Inc. All rights reserved.},
  author       = {Astrom-Olsson, Karin and Li, Lizhen and Olofsson, Charlotta S. and Boren, Jan and Öhlin, Hans and Grip, Lars},
  issn         = {1090-2104},
  keyword      = {HL-1 cells,FKBP12/FKBP12.6,Viability,Ca2+},
  language     = {eng},
  number       = {4},
  pages        = {732--738},
  publisher    = {Elsevier},
  series       = {Biochemical and Biophysical Research Communications},
  title        = {Impact of hypoxia, simulated ischemia and reperfusion in HL-1 cells on the expression of FKBP12/FKBP12.6 and intracellular calcium dynamics},
  url          = {http://dx.doi.org/10.1016/j.bbrc.2012.05.071},
  volume       = {422},
  year         = {2012},
}