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Background In heart failure (HF) both Ca2+/Calmodulin-dependent protein-kinase II (CaMKII) and late sodium current (INaL) are known to contribute to arrhythmogenesis as they contribute to action-potential (AP) prolongation and the occurrence of early- (EADs) and delayed afterdepolarizations (DADs). Further, augmented CaMKII and INaL maintain a vicious cycle as they both can activate each other. We recently found that the sodium channel isoform NaV1.8 is upregulated in HF and hypertrophy and that it is involved in INaL-generation. In the current study we investigated the effects of NaV1.8-knock-out (KO) on HF-progression and arrhythmogenesis in a CaMKII-overexpressing HF mouse model. Methods/Results CaMKII overexpressing mice (CaMKII+/T) were crossbred with NaV1.8-KO mice (SCN10A−/−). To our surprise knock-out of NaV1.8 in CaMKII+/T mice (SCN10A−/−/CaMKII+/T) significantly improved survival (median survival 103 days vs 74.5 CaMKII+/T, p<0.01). CaMKII+/T mice exhibited a strong HF phenotype compared to WT with increased heart-weight to tibia length ratio as well as reduced ejection fraction and left-ventricular end-diastolic diameter obtained by echocardiography. However, these structural parameters did not differ between SCN10A−/−/CaMKII+/T and CaMKII+/T. Therefore, cellular electrophysiology experiments were performed in isolated cardiomyocytes for a better understanding of the observed improvement in survival. INaL, measured by patch-clamp technique, was significantly augmented in CaMKII+/T vs WT and SCN10A−/−, while SCN10A−/−/CaMKII+/T showed significantly less INaL than CaMKII+/T alone. Further, AP-duration (APD) was significantly reduced in SCN10A−/−/CaMKII+/T vs CaMKII+/T while AP-amplitude, resting membrane-potential and upstroke velocity (dv/dtmax) remained unchanged. In addition, the occurrence of afterdepolarizations was significantly lower in SCN10A−/−/ CaMKII+/T vs CaMKII+/T. Confocal microscopy using the dye Fluo-4AM was performed and significantly less diastolic Ca2+-waves occurred in SCN10A−/−/CaMKII+/T compared to CaMKII+/T. In order to analyze an organ-specific SCN10A-KO, we generated homozygous SCN10A-KO lines of induced pluripotent stem cells by using CRISPR/Cas9 technology. 2-month old iPSC-cardiomyocytes lacking NaV1.8 were treated with low dose isoprenaline and showed significantly less INaL, thereby serving as a final proof of the relevant role of this Na+-channel on INaL-generation in the cardiomyocyte. Conclusion We found a survival benefit by selective knock-out of the neuronal sodium channel isoform NaV1.8 in a proarrhythmic HF mouse model with augmented CaMKII expression. However, in our model NaV1.8-knock-out showed no effects on HF progression, while cellular proarrhythmic triggers were attenuated. Taken together with our findings in IPS-cardiomyocytes treated with the CRSIPR/Cas9 technology NaV1.8 plays a significant role for the generation of INaL and cellular arrhythmogenic triggers in the cardiomyocyte. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Deutsche Stiftung für Herzforschung

Background Cardiac pathologies like hypertrophy and heart failure are known to be associated with proarrhythmogenic triggers like early- (EADs) and delayed afterdepolarizations (DADs) that can be partly attributed to an augmentation of late sodium current (INaL). Enhanced INaL is closely connected with increased activity of Ca2+/calmodulin dependent-kinase II (CaMKII) in pathology as it is enhanced by CaMKII on the one hand but can also indirectly increase CaMKII-activity on the other. We recently found neuronal sodium channel NaV1.8 to be involved in INaL-augmentation in heart failure and cardiac hypertrophy. Here, we studied possible antiarrhythmic effects of NaV1.8-inhibition in a transgenic mouse model with enhanced CaMKII-expression by selectively knocking out NaV1.8. Methods/Results To investigate antiarrhythmic effects of NaV1.8-depletion in-vivo and in-vitro we crossbred CaMKII-transgenic mice (CaMKII+/T) with NaV1.8-knock-out mice (SCN10A−/−). Surprisingly, CaMKII+/T-mice lacking NaV1.8 (CaMKII+/T & SCN10A−/−) showed a significantly improved survival compared to CaMKII+/T alone (97.5 vs 72.0 days, p<0.05). Heart weight to tibia length ratio was significantly increased in CaMKII+/T-mice compared to wild-type, without any differences between CaMKII+/T and CaMKII+/T & SCN10A−/−. To investigate the underlying mechanisms out of this observation we isolated single cardiomyocytes and performed patch-clamp experiments as well as confocal microscopy to measure Ca2+-transients and diastolic Ca2+-waves. INaL-integral was significantly smaller in cardiomyocytes from CaMKII+/T & SCN10A−/−-mice compared to CaMKII+/T alone. During action potential recordings, significantly less afterdepolarizations occurred in CaMKII+/T & SCN10A−/− compared to cardiomyocytes from CaMKII+/T -mice (16.7/min vs 34.9/min, p<0.05). There was a trend of less cells exhibiting diastolic Ca2+-waves in Ca2+-measurements from CaMKII+/T & SCN10A−/− compared to CaMKII+/T (15% vs 25%, p=0.09). As some cells showed more than one event, we calculated the frequency of Ca2+-waves and found a significant reduction of Ca2+-waves in CaMKII+/T & SCN10A−/− vs. CaMKII+/T (22.8/min vs 43.0/min, p<0.05). Moreover, the time to the first event was significantly longer in CaMKII+/T & SCN10A−/−. Ca2+-transient amplitude (F/F0) was significantly lower in CaMKII+/T compared to CaMKII+/T & SCN10A−/− (4.6 vs. 5.3, p=0.05). Further, Ca2+-extrusion from the cytosol was significantly faster in CaMKII+/T & SCN10A−/−. Conclusion Our data demonstrates, that inhibition of INaL by targeting NaV1.8 has a potent antiarrhythmic potential as we found a reduction of EADs, DADs and diastolic Ca2+-waves in CaMKII+/T & SCN10A−/−-cardiomyocytes. This antiarrhythmic potential appears to be potent enough to improve survival and to rescue the proarrhythmogenic phenotype of CaMKII-overexpression. However, further in-vivo experiments are necessary to investigate NaV1.8-inhibition for a possible therapeutic approach.

Introduction Patients with primary and secondary adrenal insufficiency (PAI/ SAI) are usually treated with glucocorticoid replacement therapy which fails to mimic the circadian rhythm of cortisol secretion thereby resulting in temporary hypo- and hypercortisolism. The unphysiological replacement is associated with impairment in cognitive function. Dual-release hydrocortisone (DR-HC) resembles the daily normal cortisol profile which improves metabolic parameters and quality of life but currently little is known about its impact on cognitive function. Material and Methods Twenty adults with adrenal insufficiency (12 PAI, 8 SAI) treated with DR-HC underwent 10 neuropsychological tests, evaluating intellectual abilities, mindset, memory, executive functioning, attention and alertness. Furthermore, demographic data, quality of life, symptoms of depression and quality of sleep were evaluated by well-established questionnaires (AddiQoL, SF-36, Beck Depression Inventory, Pittsburgh sleep quality index). Patients were split into 2 groups with respect to diagnosis (PAI vs. SAI) and to dosage (≤20mg/d vs. >20 mg/d, defined by median split). Because of use of standardized tests all patients could be compared to healthy controls. Moreover eighteen DR-HC treated adults were compared with eighteen adults on conventional-HC treatment matched for age, sex, education and diagnosis. Results Compared to standard values patients on DR-HC reached improved scores in intellectual abilities (p=0.000) and memory (p=0.001) and impaired scores in alertness (p=0.000). With respect to diagnosis patients with PAI performed better on intellectual abilities (IQ 121.3 vs. 103.8; p=0.038) and on executive functioning (79.2% vs. 45.0%; p=0.026) and reported a longer time to fall asleep (36 min. vs. 12 min.; p=0.026) than patients with SAI. Regarding DR-HC dosage, there was no significant difference in cognitive function. Patients on high dose reported a better subjective quality of sleep (p=0.028) than patients on low dose. In comparison to conventional-HC treatment, patients with DR-HC tended to show better results in executive functioning (59.2% vs. 66.1%; p=0.099). We found no further significant differences between both treatment modalities. Conclusion Patients with PAI reached better results in several cognitive functions and had a worse quality of sleep than patients with SAI. Our data suggest a positive impact of DR-HC on quality of sleep. DR-HC may be better for executive functioning than conventional-HC.