Cardioprotective stress response in the human fetal heart.
OBJECTIVE
We propose that the fetal heart is highly resilient to hypoxic stress. Our objective was to elucidate the human fetal gene expression profile in response to simulated ischemia and reperfusion to identify molecular targets that account for the innate cardioprotection exhibited by the fetal phenotype.


METHODS
Primary cultures of human fetal cardiac myocytes (gestational age, 15-20 weeks) were exposed to simulated ischemia and reperfusion in vitro by using a simulated ischemic buffer under anoxic conditions. Total RNA from treated and baseline cells were isolated, reverse transcribed, and labeled with Cy3 or Cy5 and hybridized to a human cDNA microarray for expression analysis. This analysis revealed a highly significant (false discovery rate, <3%) suppression of interleukin 6 transcript levels during the reperfusion phase confirmed by means of quantitative polymerase chain reaction (0.25 +/- 0.11-fold). Interleukin 6 signaling during ischemia and reperfusion was assessed at the protein expression level by means of Western measurements of interleukin 6 receptor, the signaling subunit of the interleukin 6 receptor complex (gp130), and signal transducer of activated transcription 3. Posttranslational changes in the protein kinase B signaling pathway were determined on the basis of the phosphorylation status of protein kinase B, mitogen-activated protein kinase, and glycogen synthase kinase 3beta. The effect of suppression of a prohypertrophic kinase, integrin-linked kinase, with short-interfering RNA was determined in an ischemia and reperfusion-stressed neonatal rat cardiac myocyte model. Endogenous secretion of interleukin 6 protein in culture supernatants was measured by enzyme-linked immunosorbent assay.


RESULTS
Human fetal cardiac myocytes exhibited a significantly lower rate of apoptosis induction during ischemia and reperfusion and after exposure to staurosporine and recombinant interleukin 6 compared with that observed in neonatal rat cardiac myocytes ( P < .05 for all comparisons, analysis of variance). Exposure to exogenously added recombinant interleukin 6 increased the apoptotic rate in both rat and human fetal cardiac myocytes ( P < .05). Short-interfering RNA-mediated suppression of integrin-linked kinase, a prohypertrophy upstream kinase regulating protein kinase B and glycogen synthase kinase 3beta phosphorylation, was cytoprotective against ischemia and reperfusion-induced apoptosis in neonatal rat cardiac myocytes ( P < .05).


CONCLUSIONS
Human fetal cardiac myocytes exhibit a uniquely adaptive transcriptional response to ischemia and reperfusion that is associated with an apoptosis-resistant phenotype. The stress-inducible fetal cardiac myocyte gene repertoire is a useful platform for identification of targets relevant to the mitigation of cardiac ischemic injury and highlights a novel avenue involving interleukin 6 modulation for preventing the cardiac myocyte injury associated with ischemia and reperfusion.
Coles
John G
JG
Department of Cardiovascular Surgery, Hospital for Sick Children, University of Toronto, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. john.coles@sickkids.ca
Boscarino
Cathy
C
Takahashi
Mark
M
Grant
Diane
D
Chang
Astra
A
Ritter
Julia
J
Dai
Xiaojing
X
Du
Changqing
C
Musso
Gabriel
G
Yamabi
Hideaki
H
Goncalves
Jason
J
Kumar
Ashu Sunny
AS
Woodgett
James
J
Lu
Huanzhang
H
Hannigan
Gregory
G
D000222:Adaptation, Physiological; D000367:Age Factors; D000818:Animals; D017209:Apoptosis / Q000235:genetics; D015153:Blotting, Western; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005315:Fetal Diseases / Q000196:embryology / Q000235:genetics / Q000378:metabolism* / Q000517:prevention & control; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D038362:Glycogen Synthase Kinase 3 / Q000502:physiology; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D015850:Interleukin-6 / Q000032:analysis / Q000502:physiology; D048369:MAP Kinase Kinase 1 / Q000502:physiology; D015428:Myocardial Reperfusion Injury / Q000196:embryology / Q000235:genetics / Q000378:metabolism* / Q000517:prevention & control; D032383:Myocytes, Cardiac / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D010084:Oxidation-Reduction; D010641:Phenotype; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D015398:Signal Transduction / Q000502:physiology; D015533:Transcriptional Activation / Q000502:physiology
D015850:Interleukin-6; D011518:Proto-Oncogene Proteins; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; D048369:MAP Kinase Kinase 1