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

Background: Acute stimulation of the late sodium current (I _NaL ) as pharmacologically induced by Anemonia toxin II (ATX-II) results in Na ⁺ -dependent Ca ²⁺ overload and enhanced formation of reactive oxygen species (ROS). This is accompanied by an acute increase in the amplitude of the systolic Ca ²⁺ transient. Ca ²⁺ transient amplitude is determined by L-type Ca ²⁺ -mediated transsarcolemmal Ca ²⁺ influx (I _Ca ) into the cytosol and by systolic Ca ²⁺ release from the sarcoplasmic reticulum (SR). Type-1 protein kinase A (PKARIα) becomes activated upon increased ROS and is capable of stimulating I _Ca , thereby sustaining the amplitude of the systolic Ca ²⁺ transient upon oxidative stress.
Objectives: We aimed to investigate whether the increase of the systolic Ca ²⁺ transient as acutely induced by I _NaL (by ATX-II) may involve stimulation of I _Ca 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 _NaL in freshly isolated ventricular myocytes from KI and wild-type (WT) control mice. Epifluorescence and confocal imaging were used to assess intracellular Ca ²⁺ handling and ROS formation. A ruptured-patch whole-cell voltage-clamp was used to measure I _NaL and I _Ca . The impact of acutely enhanced I _NaL on RIα dimer formation and PKA target structures was studied using Western blot analysis.
Results: ATX-II increased I _NaL 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 ²⁺ handling in terms of increased Ca ²⁺ transient amplitudes and elevated SR Ca ²⁺ load was equally present in KI and WT cells. Likewise, cellular arrhythmias as approximated by non-triggered Ca ²⁺ elevations during Ca ²⁺ transient decay and by diastolic SR Ca ²⁺ -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 _Ca 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 _NaL are associated with elevated ROS, but do not involve oxidation of PKARIα.
Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
(© 2024 Gissibl, Stengel, Tarnowski, Maier, Wagner, Feder and Sag.)