The inotropic and arrhythmogenic effects of acutely increased late I are associated with elevated ROS but not oxidation of PKARIα.

BACKGROUND

Acute stimulation of the late sodium current (I) as pharmacologically induced by Anemonia toxin II (ATX-II) results in Na-dependent Ca overload and enhanced formation of reactive oxygen species (ROS). This is accompanied by an acute increase in the amplitude of the systolic Ca transient. Ca transient amplitude is determined by L-type Ca-mediated transsarcolemmal Ca influx (I) into the cytosol and by systolic Ca release from the sarcoplasmic reticulum (SR). Type-1 protein kinase A (PKARIα) becomes activated upon increased ROS and is capable of stimulating I, thereby sustaining the amplitude of the systolic Ca transient upon oxidative stress.

OBJECTIVES

We aimed to investigate whether the increase of the systolic Ca transient as acutely induced by I (by ATX-II) may involve stimulation of I through oxidized PKARIα.

METHODS

We used a transgenic mouse model in which PKARIα was made resistant to oxidative activation by homozygous knock-in replacement of redox-sensitive Cysteine 17 with Serine within the regulatory subunits of PKARIα (KI). ATX-II (at 1 nmol/L) was used to acutely enhance I in freshly isolated ventricular myocytes from KI and wild-type (WT) control mice. Epifluorescence and confocal imaging were used to assess intracellular Ca handling and ROS formation. A ruptured-patch whole-cell voltage-clamp was used to measure I and I. The impact of acutely enhanced I on RIα dimer formation and PKA target structures was studied using Western blot analysis.

RESULTS

ATX-II increased I to a similar extent in KI and WT cells, which was associated with significant cytosolic and mitochondrial ROS formation in both genotypes. Acutely activated Ca handling in terms of increased Ca transient amplitudes and elevated SR Ca load was equally present in KI and WT cells. Likewise, cellular arrhythmias as approximated by non-triggered Ca elevations during Ca transient decay and by diastolic SR Ca-spark frequency occurred in a comparable manner in both genotypes. Most importantly and in contrast to our initial hypothesis, ATX-II did not alter the magnitude or inactivation kinetics of I in neither WT nor KI cells and did not result in PKARIα dimerization (i.e., oxidation) despite a clear prooxidant intracellular environment.

CONCLUSIONS

The inotropic and arrhythmogenic effects of acutely increased I are associated with elevated ROS, but do not involve oxidation of PKARIα.