Protein kinase/phosphatase balance mediates the effects of increased late sodium current on ventricular calcium cycling.

Increased late sodium current (late INa) is an important arrhythmogenic trigger in cardiac disease. It prolongs cardiac action potential and leads to an increased SR Ca2+ leak. This study investigates the contribution of Ca2+/Calmodulin-dependent kinase II (CaMKII), protein kinase A (PKA) and conversely acting protein phosphatases 1 and 2A (PP1, PP2A) to this subcellular crosstalk. Augmentation of late INa (ATX-II) in murine cardiomyocytes led to an increase of diastolic Ca2+ spark frequency and amplitudes of Ca2+ transients but did not affect SR Ca2+ load. Interestingly, inhibition of both, CaMKII and PKA, attenuated the late INa-dependent induction of the SR Ca2+ leak. PKA inhibition additionally reduced the amplitudes of systolic Ca2+ transients. FRET-measurements revealed increased levels of cAMP upon late INa augmentation, which could be prevented by simultaneous inhibition of Na+/Ca2+-exchanger (NCX) suggesting that PKA is activated by Ca2+-dependent cAMP-production. Whereas inhibition of PP2A showed no effect on late INa-dependent alterations of Ca2+ cycling, additional inhibition of PP1 further increased the SR Ca2+ leak. In line with this, selective activation of PP1 yielded a strong reduction of the late INa-induced SR Ca2+ leak and did not affect systolic Ca2+ release. This study indicates that phosphatase/kinase-balance is perturbed upon increased Na+ influx leading to disruption of ventricular Ca2+ cycling via CaMKII- and PKA-dependent pathways. Importantly, an activation of PP1 at RyR2 may represent a promising new toehold to counteract pathologically increased kinase activity.

Antiarrhythmic effects of dantrolene in human diseased cardiomyocytes.

BACKGROUND: Cardiac type 2 ryanodine receptors (RyR2s) play a pivotal role in cellular electrophysiology and contractility. Increased RyR2-mediated diastolic sarcoplasmic reticulum (SR) Ca2+ release is linked to heart failure (HF) and arrhythmias. Dantrolene, a drug used for the treatment of malignant hyperthermia, is known to stabilize RyRs in skeletal muscle. OBJECTIVE: The purpose of this study was to investigate the effects of dantrolene on arrhythmogenic triggers and contractile function in human atrial fibrillation (AF) and HF cardiomyocytes (CM). METHODS: Human CM were isolated from either patients with HF (ventricular) or patients with AF (atrial), and Ca2+ imaging, patch-clamp, or muscle strip experiments were performed. RESULTS: After exposure to dantrolene, human atrial AF and left ventricular HF CM showed significant reductions in proarrhythmic SR Ca2+ spark frequency and diastolic SR Ca2+ leak. Moreover, dantrolene decreased the frequency of Ca2+ waves and spontaneous Ca2+ transients in HF CM. Patch-clamp experiments revealed that dantrolene significantly suppressed delayed afterdepolarizations in HF and AF CM. Importantly, dantrolene had no effect on action potential duration in AF or in HF CM. In addition, dantrolene had neutral effects on contractile force of human isometrically twitching ventricular HF trabeculae. CONCLUSION: Our study showed that dantrolene beneficially influenced disrupted SR Ca2+ homeostasis in human HF and AF CM. Cellular arrhythmogenic triggers were potently suppressed by dantrolene, whereas action potential duration and contractility were not affected. As a clinically approved drug for the treatment of malignant hyperthermia, dantrolene may be a potential antiarrhythmic drug for patients with rhythm disorders and merits further clinical investigation.