Progression of excitation-contraction coupling defects in doxorubicin cardiotoxicity.

Cardiac failure is a common complication in cancer survivors treated with anthracyclines. Here we followed up cardiac function and excitation-contraction (EC) coupling in an in vivo doxorubicin (Dox) treated mice model (iv, total dose of 10 mg/Kg divided once every three days). Cardiac function was evaluated by echocardiography at 2, 6 and 15 weeks after the last injection. While normal at 2 and 6 weeks, ejection fraction was significantly reduced at 15 weeks. In order to evaluate the underlying mechanisms, we measured [Ca] transients by confocal microscopy and action potentials (AP) by patch-clamp technique in cardiomyocytes isolated at these times. Three phases were observed: 1/depression and slowing of the [Ca] transients at 2 weeks after treatment, with occurrence of proarrhythmogenic Ca waves, 2/compensatory state at 6 weeks, and 3/depression on [Ca] transients and cell contraction at 15 weeks, concomitant with in-vivo defects. These [Ca] transient alterations were observed without cellular hypertrophy or AP prolongation and mirrored the sarcoplasmic reticulum (SR) Ca load variations. At the molecular level, this was associated with a decrease in the sarcoplasmic reticulum Ca ATPase (SERCA2a) expression and enhanced RyR2 phosphorylation at the protein kinase A (PKA, pS2808) site (2 and 15 weeks). RyR2 phosphorylation at the Ca/calmodulin dependent protein kinase II (CaMKII, pS2814) site was enhanced only at 2 weeks, coinciding with the higher incidence of proarrhythmogenic Ca waves. Our study highlighted, for the first time, the progression of Dox treatment-induced alterations in Ca handling and identified key components of the underlying Dox cardiotoxicity. These findings should be helpful to understand the early-, intermediate-, and late- cardiotoxicity already recorded in clinic in order to prevent or treat at the subclinical level.