Cardiomyoblast apoptosis induced by insulin-like growth factor (IGF)-I resistance is IGF-II dependent and synergistically enhanced by angiotensin II.
OBJECTIVE
This study explores the synergistic effect of cardiomyoblast apoptosis induced by angiotensin II (Ang II) and Insulin-like growth factor (IGF)-I resistance, and elucidates the role of IGF-II via IGF-II receptor (R) and calcineurin pathways in apoptosis induced by Ang II and IGF-I resistance.


METHODS
Apoptosis of cultured cardiomyoblast H9c2 cells was assessed by DNA fragmentation on agarose gel electrophoresis, nuclear condensation stained with DAPI, and Western blot analysis of pro-apoptotic Bad and cytochrome c in various combinations of control, Ang II, antisense IGF (I or II), IGF (I or II) antibody, IGF (I or II) receptor (R) antibody, or calcineurin inhibitor (Cyclosporine A, (CsA)).


RESULTS
We found the following: (1) The combination of Ang II and IGF-I deficiencies had a synergistic effect on apoptosis, confirmed by DNA fragmentation, nuclei condensation, and increases in such pro-apoptotic proteins as Bad, cytochrome c, caspase 9, and caspase 3 in H9c2 cells. (2) IGF-II and IGF-IIR protein products were increased by antisense IGF-I and IGF-I resistance, but these IGF-II protein products were not affected by sense IGF-I and non-specific antibody IgG in H9c2 cells. (3) The alteration of Bad protein level and the release of cytochrome c, both induced by treatments containing combinations of Ang II and antisense IGF-I, IGF-I antibody or IGF-IR antibody, were inhibited by IGF-II antibody. (4) DNA fragmentation, Bad, and cytochrome c which was induced by treatments combining IGF-IR antibody with Ang II or combining IGF-IR antibody with IGF-II were remarkably attenuated by CsA.


CONCLUSIONS
IGF-I deficiency and/or IGF-IR resistance induced apoptosis in cardiomyoblast cells. The apoptosis, which might have been caused by the upregulation of IGF-II and IGF-IIR genes possibly activated the downstream calcineurin pathway, was synergistically augmented by Ang II.
Kuo
Wei-Wen
WW
Department of Biological Science and Technology, China Medical University, No. 91 Hsueh-Shih Road, Taichung 404, Taiwan.
Liu
Chung-Jung
CJ
Chen
Li-Ming
LM
Wu
Chieh-Hsi
CH
Chu
Chun-Hsien
CH
Liu
Jer-Yuh
JY
Lu
Min-Chi
MC
Lin
James A
JA
Lee
Shin-Da
SD
Huang
Chih-Yang
CY
D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D019703:Calcineurin / Q000502:physiology; D065095:Calcineurin Inhibitors; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D016572:Cyclosporine / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004357:Drug Synergism; D020287:In Situ Nick-End Labeling; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D007335:Insulin-Like Growth Factor II / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008954:Models, Biological; D032386:Myoblasts, Cardiac / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000276:immunology / Q000502:physiology; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D016559:Tacrolimus / Q000494:pharmacology; D014162:Transfection; D051025:bcl-Associated Death Protein / Q000378:metabolism
D000906:Antibodies; C494821:Bad protein, rat; D065095:Calcineurin Inhibitors; D016376:Oligonucleotides, Antisense; D017527:Receptor, IGF Type 2; D051025:bcl-Associated Death Protein; D000804:Angiotensin II; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D016572:Cyclosporine; D045304:Cytochromes c; D017526:Receptor, IGF Type 1; D051057:Proto-Oncogene Proteins c-akt; D019703:Calcineurin; C505410:Casp3 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; D016559:Tacrolimus