Korean J Anesthesiol.  2006 Jun;50(6):S48-S56. 10.4097/kjae.2006.50.6.S48.

Bupivacaine-induced Apoptosis in the Primary Cultured Cardiomyocytes via p38 MAPKs

• Kim HJ
• Sung SR
• Seo KS
• Lim SW
• Yoon TG

• KMID: 1862264
• DOI: http://doi.org/10.4097/kjae.2006.50.6.S48

Abstract

BACKGROUND: It is known that bupivacaine induce cell death in several immortalized cells. However, there is no report concerning bupivacaine-induced cell death in the primary cultured cardiomyocytes. We compared the direct cytotoxicity of local anesthetics in cardiomyocytes. Furthermore, the mechanisms of cell death were evaluated.
METHODS
The myocardial cells of rat pups were cultured 3 days after seeding. The methyltetrazolium (MTT) assay was employed to quantify differences in cellular viability. To confirm apoptosis, Hoechst-propidium iodide staining, DNA fragmentation by electrophoresis and western blot analysis were performed. And to examine the mechanisms of cell death, intracellular calcium and expression levels of mitogen-activated protein kinases (MAPKs) family members were evaluated.
RESULTS
Among the local anesthetics under 1 mM concentration for 18 h, only bupivacaine significantly decreased the MTT activity (P < 0.001). Bupivacaine induced cell death in a dose-responsive and time dependent manner. Cell death showed apoptotic characteristics, such as DNA fragmentation, chromatin condensation, decrease of precursor caspase-3 protein level, increased cleaved PARP, and cytochrome C release into the cytoplasm. Bupivacaine phosphorylated three major MAPKs, i.e. extracellular signal-regulated kinases (ERKs), p38 kinase and c-Jun N-terminal kinases (JNKs) stress-activated protein kinases. Administration of ERK inhibitor increase cell death, whereas inhibitors of p38 kinase and JNK decreased cell death (P < 0.05). In addition, the intracellular calcium level was approximately 4 times higher after the bupivacaine treatment (P < 0.001), which was inhibited by calcium chelators (P < 0.001). Calcium chelators inhibited expression of MAPKs.
CONCLUSIONS
In bupivacaine-induced apoptosis in cardiomyocytes, intracellular calcium increase and MAPKs family plays important roles.

Keyword

apoptosis; bupivacaine; calcium; cardiomyocyte; levobupivacaine; mitogen-activated protein kinases

MeSH Terms

Anesthetics, Local
Animals
Apoptosis*
Blotting, Western
Bupivacaine
Calcium
Caspase 3
Cell Death
Chelating Agents
Chromatin
Cytochromes c
Cytoplasm
DNA Fragmentation
Electrophoresis
Extracellular Signal-Regulated MAP Kinases
Humans
Mitogen-Activated Protein Kinases
Myocytes, Cardiac*
p38 Mitogen-Activated Protein Kinases*
Phosphotransferases
Protein Kinases
Rats
Anesthetics, Local
Bupivacaine
Calcium
Caspase 3
Chelating Agents
Chromatin
Cytochromes c
Extracellular Signal-Regulated MAP Kinases
Mitogen-Activated Protein Kinases
Phosphotransferases
Protein Kinases
p38 Mitogen-Activated Protein Kinases