Anti-erbB2 causes mitochondrialdysfunction and oxidant stress in cardiac myocytes

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Introduction: Since the introduction of anthracyclines, cardiotoxicity has been recognized as a major side effect of chemotherapy, often limiting needed treatment. A monoclonal antibody inhibiting erbB2 is a potent chemotherapeutic agent but is associated with cardiac toxicity. Here we investigate the effects of anti-erbB2 antibody on mitochondrial function and cardiomyocyte survival. Methods and Results: Primary cultures of neonatal and adult rat ventricular myocytes were exposed to anti-erbB2 antibody (anti-erbB2 Ab 5-7.5 μg/ml) for up to 24 hours. Cell viability, mitochondrial function, and apoptosis were measured using multiple complementary techniques. ErbB2 inhibition was associated with a dramatic increase in expression of the Bcl-2 family protein, Bcl-xS_, and decreased levels of Bcl-xL. There was a time dependent increase in mitochondrial translocation and oligomerization of Bax as indicated by subcellular fractionation and BMH crosslinking. Bax oligomerization was associated with cytochrome c release and caspase activation. This alteration in Bcl-2 family signaling induced mitochondrial dysfunction evident as a loss of mitochondrial membrane potential (Ψ) as measured by fluorescence flow cytometry of mitotracker stained NRVM (76.9 ± 2.4 vs 51.7 ± 0.07 p< 0.05 N=4), a 35% decline in ATP levels (p< 0.05) as measured by luciferin-luciferase bioluminescence, a loss of redox capacity as measured by MTT (0.72 ± 0.036 vs 0.64 ± 0 .017 p< 0.01) and an increase in oxidant stress (0.079 ± 0.005 vs 0.066 ± 0.002 N=3 P= 0.0501) as measured by 5,6 dichlorodihydrofluorescein diacetate luminescence. Restoration of Bcl-xL levels through TAT-mediated protein transduction prevented the decline inΨ, MTT activity and cytosolic ATP. Anti-erbB2 exposure resulted in a minimal increase in apoptosis as measured by TUNEL (6.5 ± 0.7 vs 3.1 ± 0.4%. p< 0.05) and propidium iodide flow cytometry ( 8.3 ± 0.9 vs 4.5 ± 0.6 % p< 0.01) and no discernible difference in DNA laddering. Conclusion: We conclude anti-erbB2 induces mitochondrial dysfunction through modulation of Bcl-xL and Bcl-xS and leads to activation of the mitochondrial apoptotic signaling pathway in the absence of substantial apoptosis. While the mechanism underlying this “interrupted apoptosis” is not yet clear, we hypothesize that this state,in which cardiomyocytes may be functionally impaired, and more susceptibile to additional apoptotic stimuli, may offer opportunities for reverse remodeling and therapeutic intervention. Future studies will be necessary to define the contribution of these mechanisms to the cardiac dysfunction seen with erbB2 in in vivo models or in clinical settings.