Yonsei Med J.  2010 Mar;51(2):187-196. 10.3349/ymj.2010.51.2.187.

Cardioprotection Via Modulation of Calcium Homeostasis by Thiopental in Hypoxia-Reoxygenated Neonatal Rat Cardiomyocytes

• Kim HS
• Hwang KC
• Park WK

• KMID: 1126016
• DOI: http://doi.org/10.3349/ymj.2010.51.2.187

Abstract

PURPOSE
Ca2+ homeostasis plays an important role in myocardial cell injury induced by hypoxia-reoxygenation, and prevention of intracellular Ca2+ overload is key to cardioprotection. Even though thiopental is a frequently used anesthetic agent, little is known about its cardioprotective effects, particulary in association with Ca2+ homeostasis. We investigated whether thiopental protects cardiomyocytes against hypoxia-reoxygenation injury by regulating Ca2+ homeostasis.
MATERIALS AND METHODS
Neonatal rat cardiomyocytes were isolated. Cardiomyocytes were exposed to different concentrations of thiopental and immediately replaced in the hypoxic chamber to maintain hypoxia. After 1 hour of exposure, a culture dish was transferred to the CO2 incubator and cells were incubated at 37degrees C for 5 hours. At the end of the experiments, the authors assessed cell protection using immunoblot analysis and caspase activity. The mRNA of genes involved in Ca2+ homeostasis, mitochondrial membrane potential, and cellular Ca2+ levels were examined.
RESULTS
In thiopental-treated cardiomyocytes, there was a decrease in expression of the proapoptotic protein Bax, caspase-3 activation, and intracellular Ca2+ content. In addition, both enhancement of anti-apoptotic protein Bcl-2 and activation of Erk concerned with survival were shown. Furthermore, thiopental attenuated alterations of genes involving Ca2+ regulation and significantly modulated abnormal changes of NCX and SERCA2a genes in hypoxia-reoxygenated neonatal cardiomyocytes. Thiopental suppressed disruption of mitochondrial membrane potential (Delta Psi m) induced by hypoxia-reoxygenation.
CONCLUSION
Thiopental is likely to modulate expression of genes that regulate Ca2+ homeostasis, which reduces apoptotic cell death and results in cardioprotection.

Keyword

Thiopental; Ca2+ homeostasis; hypoxia-reoxygenation; cardiomyocytes

MeSH Terms

Animals
Apoptosis
Calcium/*metabolism
Cell Hypoxia/*physiology
Cell Survival/drug effects
Cells, Cultured
GABA Modulators/*pharmacology
Homeostasis/drug effects
Immunoblotting
In Situ Nick-End Labeling
Membrane Potential, Mitochondrial/drug effects
Microscopy, Confocal
Myocytes, Cardiac/*drug effects/*metabolism
Rats
Rats, Sprague-Dawley
Reverse Transcriptase Polymerase Chain Reaction
Thiopental/*pharmacology

Figure

• 

  Fig. 1 Effect of thiopental on survival of hypoxia-reoxygenated cardiomyocytes. (A) Relative cell viability of hypoxia-reoxygenated neonatal rat cardiomyocytes pretreated with various concentrations of thiopental (0.1 - 500 µM). Cardiomyocytes were plated in triplicate wells in 96-well plates at a density of 1×104 per well, and pretreated with various concentrations of thiopental. Then, cardiomyocytes were subjected to a hypoxic chamber for 1 hour following reoxygenation for 5 hours. Cell viability was determined by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (B) Western blot analysis of phosphorylation of extracellular signal-regulated kinases (ERK). Each signal was quantified by scanning densitometry. Data presented as mean ± S.E.M. above 2-3 replicate measurements in 3 cell cultures (n = 7). Control: normal cardiomyocytes. H/R: hypoxia-reoxygenated cardiomyocytes. *p < 0.05, **p < 0.01.

• 

  Fig. 2 Effect of thiopental on activity of proteins related to apoptosis. (A) Effect of thiopental (50 µM) on Bcl-2 and Bax in hypoxia-reoxygenated cells. Western blot analysis of Bcl-2 and Bax. Each signal was quantified by scanning densitometry. (B) Effect of thiopental (50 µM) on apoptotic index in hypoxia-reoxygenated cells. The apoptotic index was determined by the number of positively stained apoptotic myocytes/total number of myocytes counted×100%. (C) Effect of thiopental (50 µM) on caspase-3 activity in hypoxia-reoxygenated cells. Relative caspase-3 activity was determined using the ApopTarget™ Capase-3. Data is presented as mean ± S.E.M. above 2 - 3 replicate measurements in 3 cell cultures (n = 7). Control: normal cardiomycytes. H/R: hypoxia-reoxygenated cardiomyocytes. *p < 0.05, **p < 0.01.

• 

  Fig. 3 Effect of thiopental (50 µM) on intracellular Ca2+ concentration in hypoxiareoxygenated cells. Cytosolic free Ca2+ concentration was determined with relative fluorescence intensity. Data is presented as mean ± S.E.M. above 2-3 replicate measurements in three different cell cultures (n = 5). Control: normal cardiomycytes. H/R: hypoxia-reoxygenated cardiomyocytes. *p < 0.05.

• 

  Fig. 4 Analysis of gene expressions related to Ca2+ homeostasis. mRNA expression of genes was established by separating amplification products by agarose gel electrophoresis and visualizing them by ethidium bromide staining. Values were normalized to GAPDH before calculating changes. Data is presented as mean ± S.E.M. above 2-3 replicate measurements in three different cell cultures (n = 6). Control: normal cardiomycytes. H/R: hypoxia-reoxygenated cardiomyocytes. *p < 0.05, **p < 0.01. PMCA1, sarcolemmal Ca2+ pump; SERCA2a, sarcoplasmic reticulum Ca2+-ATPase; NCX, Na+-Ca2+ exchanger; PLB, phospholamban; RyR2, ryanodine receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

• 

  Fig. 5 Effect of thiopental (50 µM) on Western blot analysis of SERCA2a and NCX. Each signal was quantified by scanning densitometry. Values were normalized to β-actin before calculating changes. Data is presented as mean ± S.E.M. above 2 - 3 replicate measurements in 3 cell cultures (n = 7). Control: normal cardiomycytes. H/R: hypoxia-reoxygenated cardiomyocytes. *p < 0.05.

• 

  Fig. 6 Effect of thiopental (50 µM) on mitochondrial membrane potential in hypoxia-reoxygenated cells. Mitochondrial membrane potential was estimated by flow cytometry. Cells were labeled with JC-1 for 20 min at 37℃ (n = 5). Hypoxia-reoxygenation increased mitochondrial membrane depolarization (middle panel), and thiopental treatment inhibited this effect (right panel). Control: normal cardiomyocytes. H/R: hypoxia-reoxygenated cardiomyocytes.

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