Activator Protein 1 and Caspase 8 Mediate Palmitate‐Induced Cardiomyocyte Apoptosis

Abstract

IntroductionLipoapoptosisof cardiomyocytes may be the basis for diabetic cardiomyopathy. Wepreviously demonstrated that p38α mitogen activated protein kinase (MAPK) mediatespalmitate (PA)‐induced cardiomyocyte apoptosis, and that specific p38α siRNAmarkedly and dose‐dependently attenuates apoptosis in cardiomyocytes treated with PA. We tested the hypothesis that p38α‐dependent pathway consists, in part, of c‐fos subunit of Activator Protein 1 (AP‐1) and caspase8, and that this p38α/AP‐1/caspase 8 cascade mediate PA‐induced cardiomyocyte apoptosis.MethodsHuman adult ventricular cardiomyocytes line (AC16 cells) were exposed to high physiological levels of PA. p38α‐dependent pathway was evaluated using p38α and c‐fos siRNA knockdown, and caspase 8 inhibitor.ResultsWith PA treatment, dose‐dependent increases in c‐fos mRNA (1.83 fold and 3.13 fold control, at 150 μM and 300 μM respectively, n=6, p<0.01), in c‐fos protein (1.00 fold and 2.67 fold, at 150 μM and 300 μM respectively, n=3, p<0.05), and a much greater increase in phospho‐c‐fos (2.64 fold and 8.94 fold, at 150 μM and 300 μM respectively, n=, p<0.01) were measured. Transcription of caspase 8 also increased (1.40 fold and 1.80 fold, at 150 μM and 300 μM respectively, n=5, p<0.05) but procaspase 8 protein level did not.p38α knockdown dose‐dependently blocked the PA‐induced increase in phospho‐c‐fos protein level(to 62%, 45%, and 30% of control (si‐control + PA 300 μM group) for 30 pmol, 60 pmol, and 120 pmol si‐p38α knockdown groups, n=5, p<0.01), but total c‐fos level did not change significantly. Interestingly, procaspase 8 level was also dose‐dependently reduced by p38α knockdown (75%, 65%, and 59% of control (si‐control + PA 300 μM group) for 30 pmol, 60 pmol, and 120 pmolsi‐p38α knockdown groups, n=5, p<0.05).With c‐fos knockdown, PA‐induced apoptosis was reduced 39% (n=3, p<0.05) 24 h post‐transfection, and by 53% after 48 h post‐transfection (n=3, p<0.01). Caspase 8 inhibition for 24 h reduced apoptosis by 39% (n=3, p<0.01).ConclusionsThis study in cardiomyocytes demonstrated that PA increases expression and phosphorylation of c‐fos subunit of AP‐1, and caspase 8 transcription, and further that p38α knockdown attenuates PA‐induced increases in c‐fos and phospho‐c‐fos, and decreases caspase 8. Translational knockdown of c‐fos attenuated PA‐induced cardiomyocyte apoptosis, as did inhibition of caspase 8. Our results support the hypothesis that PA‐induced apoptosis is mediated by p38α, AP‐1/c‐fos, and caspase8.Support or Funding InformationThis work has been funded by VA VISN 18 New Investigator Grant (to C.O.), CSR&D5I01CX000598grant (to P.R.), and VA Merit grant BLRDI01BX007080(to R.M.). The study was supported by VA Employment and Carl T. Hayden Medical Research Foundation. The contents and the views do not represent the views of the Department of Veterans Affairs or the US government.