DOX-dependently increased late INa induces CaMKII-mediated SR Ca leak that can be prevented by empagliflozin

Doxorubicin (DOX) activates the late component of cardiac Na_V1.5 (late I_Na) and induces CaMKIIδ-dependent Ca leakage from the sarcoplasmic reticulum (SR).  We hypothesized that DOX induces a vicious cycle of late I_Na-dependent Na/Ca overload activating CaMKIIδ, which in turn is followed by CaMKIIδ-dependent stimulation of Na_V1.5 through phosphorylation at Serine-571.

Murine wildtype (WT) cardiomyocytes were exposed to DOX at 10 µmol/L for 10 mins in the presence or absence of empagliflozin (EMPA, 1 µmol/L), which is known to inhibit late I_Na. In addition, cardiomyocytes from a transgenic mouse model of Na_V1.5 phosphoresistance to CaMKIδ (S571A model) were also used. Whole-cell patch clamp technique was used to measure late I_Na. Epifluorescence microscopy was used to obtain intracellular Na (SBFI) and Ca levels (Fura2-AM). Diastolic Ca spark frequency was measured by confocal microscopy (Fluo4-AM). Activation of CaMKIIδ and subsequent phosphorylation of CaMKIIδ-dependent target structures were assessed by Western blot. 

DOX acutely increased late I_Na by twofold in WT cardiomyocytes, which was associated with intracellular Na/Ca overload and impaired Ca handling in terms of depressed systolic Ca transient amplitudes. SR Ca content was depleted, likely as a consequence of the observed SR Ca leak. This was mediated by hyperphosphorylation of the RyR2 at the CaMKIIδ-specific site Serine-2814. Concomitant administration of EMPA significantly attenuated DOX-mediated induction of late I_Na and prevented SR Ca leak in the face of absent CaMKIIδ hyperactivation and unaltered phosphorylation status of Serine-2814 on RyR2. The DOX-dependent increase in late I_Na was also attenuated in DOX-treated S571A cardiomyocytes and associated with less CaMKIIδ activation and CaMKIIδ-dependent SR Ca leak. 

Our study shows that DOX induces a vicious cycle of late I_Na-dependent CaMKIIδ activation that in turn stimulates late I_Na. Pharmacological inhibition of late I_Na by EMPA as well as genetic inhibition of CaMKIIδ-dependent Na_V1.5 activation are both capable of attenuating the pathologic interplay between late I_Na and CaMKIIδ.