Background: Acute stimulation of the late sodium current (INaL) as
pharmacologically induced by Anemonia toxin II (ATX-II) results in
Na+-dependent Ca2+ overload and enhanced formation of reactive oxygen
species (ROS). This is accompanied by an acute increase in the amplitude of
the systolic Ca2+ transient. Ca2+ transient amplitude is determined by L-type
Ca2+-mediated transsarcolemmal Ca2+ influx (ICa) into the cytosol and by
systolic Ca2+ release from the sarcoplasmic reticulum (SR). Type-1 protein
kinase A (PKARIα) becomes activated upon increased ROS and is capable of
stimulating ICa, thereby sustaining the amplitude of the systolic Ca2+ transient
upon oxidative stress.
Objectives: We aimed to investigate whether the increase of the systolic Ca2+
transient as acutely induced by INaL (by ATX-II) may involve stimulation of ICa
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 redoxsensitive
Cysteine 17 with Serine within the regulatory subunits of PKARIα (KI).
ATX-II (at 1 nmol/L) was used to acutely enhance INaL in freshly isolated
ventricular myocytes from KI and wild-type (WT) control mice. Epifluorescence
and confocal imaging were used to assess intracellular Ca2+ handling and ROS
formation. A ruptured-patch whole-cell voltage-clamp was used to measure
INaL and ICa. The impact of acutely enhanced INaL on RIα dimer formation and
PKA target structures was studied using Western blot analysis.
Results: ATX-II increased INaL to a similar extent in KI and WT cells, which was
associated with significant cytosolic and mitochondrial ROS formation in both
genotypes. Acutely activated Ca2+ handling in terms of increased Ca2+ transient
amplitudes and elevated SR Ca2+ load was equally present in KI and WT cells.
Likewise, cellular arrhythmias as approximated by non-triggered Ca2+ elevations
during Ca2+ transient decay and by diastolic SR Ca2+-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 ICa 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 INaL
are associated with elevated ROS, but do not involve oxidation of PKARIα.