Aims To determine the characteristics of the late Na current (INaL) and its arrhythmogenic potential in the progression of pressure-induced heart disease. Methods and Results Transverse aortic constriction (TAC) was used to induce pressure overload in mice. After one week the hearts developed isolated hypertrophy with preserved systolic contractility. In patch-clamp experiments both, INaL and the action potential duration (APD90) were unchanged. In contrast, after five weeks animals developed heart failure with prolonged APDs and the slowed INaL decay time which could be normalized by addition of the INaL inhibitor ranolazine (Ran) or by the Ca/calmodulin-dependent protein kinase II (CaMKII) inhibitor AIP. Accordingly the APD90 could be significantly abbreviated by Ran, tetrodotoxin and the CaMKII inhibitor AIP. Isoproterenol increased the number of delayed afterdepolarizations (DAD) in myocytes from failing but not sham hearts. Application of either Ran or AIP prevented the occurrence of DADs. Moreover, the incidence of triggered activity was significantly increased in TAC myocytes and was largely prevented by Ran and AIP. Western blot analyses indicate that increased CaMKII activity and a hyperphosphorylation of the Nav1.5 at the CaMKII phosphorylation site (Ser571) paralleled our functional observations five weeks after TAC surgery. Conclusion In pressure overload-induced heart failure a CaMKII-dependent augmentation of INaL plays a crucial role in the AP prolongation and generation of cellular arrhythmogenic triggers, which cannot yet be found in early and still compensated hypertrophy. Inhibition of INaL and CaMKII exert potent antiarrhythmic effects and might therefore be of potential therapeutic interest.

Background— Sarcoplasmic reticulum (SR) Ca 2+ leak through ryanodine receptor type 2 (RyR2) dysfunction is of major pathophysiological relevance in human heart failure (HF); however, mechanisms underlying progressive RyR2 dysregulation from cardiac hypertrophy to HF are still controversial. Methods and Results— We investigated healthy control myocardium (n=5) and myocardium from patients with compensated hypertrophy (n=25) and HF (n=32). In hypertrophy, Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and protein kinase A (PKA) both phosphorylated RyR2 at levels that were not different from healthy myocardium. Accordingly, inhibitors of these kinases reduced the SR Ca 2+ leak. In HF, however, the SR Ca 2+ leak was nearly doubled compared with hypertrophy, which led to reduced systolic Ca 2+ transients, a depletion of SR Ca 2+ storage and elevated diastolic Ca 2+ levels. This was accompanied by a significantly increased CaMKII-dependent phosphorylation of RyR2. In contrast, PKA-dependent RyR2 phosphorylation was not increased in HF and was independent of previous β-blocker treatment. In HF, CaMKII inhibition but not inhibition of PKA yielded a reduction of the SR Ca 2+ leak. Moreover, PKA inhibition further reduced SR Ca 2+ load and systolic Ca 2+ transients. Conclusions— In human hypertrophy, both CaMKII and PKA functionally regulate RyR2 and may induce SR Ca 2+ leak. In the transition from hypertrophy to HF, the diastolic Ca 2+ leak increases and disturbed Ca 2+ cycling occurs. This is associated with an increase in CaMKII- but not PKA-dependent RyR2 phosphorylation. CaMKII inhibition may thus reflect a promising therapeutic target for the treatment of arrhythmias and contractile dysfunction.

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AimsEnhanced cardiac late Na current (late INa) and increased sarcoplasmic reticulum (SR)-Ca2+-leak are both highly arrhythmogenic. This study seeks to identify signalling pathways interconnecting late INa and SR-Ca2+-leak in atrial cardiomyocytes (CMs).Methods and resultsIn murine atrial CMs, SR-Ca2+-leak was increased by the late INa enhancer Anemonia sulcata toxin II (ATX-II). An inhibition of Ca2+/calmodulin-dependent protein kinase II (Autocamide-2-related inhibitory peptide), protein kinase A (H89), or late INa (Ranolazine or Tetrodotoxin) all prevented ATX-II-dependent SR-Ca2+-leak. The SR-Ca2+-leak induction by ATX-II was not detected when either the Na+/Ca2+ exchanger was inhibited (KBR) or in CaMKIIδc-knockout mice. FRET measurements revealed increased cAMP levels upon ATX-II stimulation, which could be prevented by inhibition of adenylyl cyclases (ACs) 5 and 6 (NKY 80) but not by inhibition of phosphodiesterases (IBMX), suggesting PKA activation via an AC-dependent increase of cAMP levels. Western blots showed late INa-dependent hyperphosphorylation of CaMKII as well as PKA target sites at ryanodine receptor type-2 (-S2814 and -S2808) and phospholamban (-Thr17, -S16). Enhancement of late INa did not alter Ca2+-transient amplitude or SR-Ca2+-load. However, upon late INa activation and simultaneous CaMKII inhibition, Ca2+-transient amplitude and SR-Ca2+-load were increased, whereas PKA inhibition reduced Ca2+-transient amplitude and load and additionally slowed Ca2+ elimination. In atrial CMs from patients with atrial fibrillation, inhibition of late INa, CaMKII, or PKA reduced the SR-Ca2+-leak.ConclusionLate INa exerts distinct effects on Ca2+ homeostasis in atrial myocardium through activation of CaMKII and PKA. Inhibition of late INa represents a potential approach to attenuate CaMKII activation and decreases SR-Ca2+-leak in atrial rhythm disorders. The interconnection with the cAMP/PKA system further increases the antiarrhythmic potential of late INa inhibition.

AimsThe sarcoplasmic reticulum (SR) Ca 2+ leak is an important pathomechanism in heart failure (HF). It has been suggested that Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is only relevant for the induction of the SR Ca 2+ leak in non-ischaemic but not in ischaemic HF. Therefore, we investigated CaMKII and its targets as well as the functional effects of CaMKII inhibition in human ischaemic cardiomyopathy (ICM, n = 37) and dilated cardiomyopathy (DCM, n = 40 Methods and resultsWestern blots showed a significantly increased expression (by 54 ± 9%) and autophosphorylation at Thr286 (by 129 ± 29%, P < 0.05 each) of CaMKII in HF compared with healthy myocardium. However, no significant difference could be detected in ICM compared with DCM as to the expression and autophosphorylation of CaMKII nor the phosphorylation of the target sites ryanodine receptor 2 (RyR2)-S2809, RyR2-S2815, and phospholamban-Thr17. Isolated human cardiomyocytes (CMs) of patients with DCM and ICM showed a similar frequency of diastolic Ca 2+ sparks (confocal microscopy) as well as of major arrhythmic events (Ca 2+ waves, spontaneous Ca 2+ transients). Despite a slightly smaller size of Ca 2+ sparks in DCM (P < 0.01), the calculated SR Ca 2+ leak [Ca 2+ spark frequecy (CaSpF) × amplitude × width × duration] did not differ between CMs of ICM vs. DCM. Importantly, CaMKII inhibition by autocamide-2-related inhibitory peptide (AIP, 1 μmol/L) reduced the SR Ca 2+ leak by ∼80% in both aetiologies (P < 0.05 each) and effectively decreased the ratio of arrhythmic cells (P < 0.05).