Knockdowns of AMPK α Subunit Isoforms Result in Differential H9c2 Cell Responses to Doxorubicin Treatment

Abstract

Doxorubicin (DOX) is an anthracycline anticancer drug which unfortunately causes severe dose-dependent dilated cardiomyopathy in some patients through poorly defined mechanisms. AMP-activated protein kinase (AMPK) is an important cellular energy regulator which plays a protective role in conditions such as ischemic heart disease and diabetic cardiomyopathy when activated. AMPK activation is believed to be similarly protective against DOX-induced cardiomyopathy. However, there has been little to no investigation into the role of the different subunits of AMPK or their individual isoforms in the cellular response to DOX. Previous studies have demonstrated subunit- and isoform-specific differences in effect with other drugs that act on or via AMPK, suggesting the possibility that certain AMPK isoforms may either mediate or attenuate the cardiotoxic effects of DOX. In the present study, we investigated the effects of knocking down isoforms of the catalytic α subunit of AMPK on DOX-induced cytotoxicity. H9c2 cardioblast cells were treated with siRNA to knock down the expression of α1, α2, or both and then exposed to DOX. Cell death was determined by propidium iodide (PI) staining that indicates necrosis and Western blot analysis of the expression levels of cleaved caspase 3 that signals apoptosis. Concurrent knockdown of both α1 and α2 isoforms dramatically reduced DOX-induced H9c2 cell death as shown by the decreased number of PI positive cells and the amount of cleaved caspase 3. These data indicate that the AMPK signaling pathway may have contributed to DOX cardiotoxicity, in stark contrast to the predominant belief that AMPK is cardioprotective. More interestingly, whereas α1 knockdown decreased DOX-induced cell death to the same degree as knocking down both α1 and α2 isoforms, α2 knockdown alone had minimal effect on DOX cytotoxicity. These results clearly demonstrate the isoform-dependent differential effects of AMPK α on H9c2 cell response to DOX treatment. Further studies are warranted to elucidate the relative importance of AMPK α, β, and γ subunits and their isoforms in either mediating or attenuating DOX cardiotoxicity.