Unveiling new antiarrhythmic mechanisms: Sacubitrilat exerts direct antiarrhythmic effects targeting NPR-C

Introduction
Sacubitril/valsartan, an angiotensin receptor neprilysin inhibitor (ARNI), is a key treatment in heart failure and significantly reduces cardiovascular mortality. Furthermore, clinical studies demonstrated that ARNI-treatment markedly reduces the incidence of ICD shocks and ventricular arrhythmias. Prior research indicated that Sacubitril (Sac) exerts a direct antiarrhythmic effect on murine and human ventricular cardiomyocytes. The mode of action of this effect remains elusive. Possible mechanisms include enhanced natriuretic peptide-signaling (ANP, BNP, CNP) and consecutively activated downstream receptor pathways (GC-A, GC-B, NPR-C). The aim of this study was to elucidate the direct antiarrhythmic effect of Sac and to identify novel antiarrhythmic toeholds. 

Methods
Ventricular Cardiomyocytes of wild-type Mice (C57BL/6N) were isolated using Langendorff-perfusion. Cardiomyocytes were treated with ATX-II to destabilize ion-homeostasis mimicking conditions in heart failure.  Treatment groups additionally received CNP (100nM), ANP (100nM), Sac (40µM) and/or NPR-C blocker osteocrin (Ocn, 100nM) and NPR-C activator cANP_4-23 (100nM). The calcium spark frequency (CaSpF) was analyzed using laser scanning confocal microscopy. Additionally systolic calcium transients, calcium transient kinetics and SR calcium load was measured using epifluorescence microscopy. To determine the impact of Sac on cGMP production we performed radioimmunoassays. Data were analyzed using GraphPad Prism. One-way ANOVA with Tukey's post hoc test was applied, and significance was set at p<0.05. 

Results
In line with prior research, ATX-II significantly enhanced diastolic CaSpF compared to control (Con vs. ATX-II 0.7 vs. 1.4 x100µm^-1s^-1; p<0.0001). This effect was abolished upon administration of Sacubitrilat (ATX-II vs. ATX-II+Sac: 0.82 vs. 0.51 x100µm^-1s^-1, p=0.014) and CNP (ATX-II vs. ATX-II+CNP: 1.4 vs. 0.87 x100µm^-1s^-1; p=0.0006). In contrast, ANP did not lower CaSpF compared to sole ATX-II treatment (p=n.s.). To evaluate, which CNP-receptor (GC-B vs. NPR-C) is responsible for reduction of CaSpF, we blocked NPR-C with Osteocrin. Upon co-incubation with Ocn effects of Sac and CNP on CaSpF were blunted (ATX-II vs. ATX-II+CNP/Ocn: 1.05 vs. 1.22 x100µm^-1s^-1, p=n.s., ATX-II vs. ATX-II+Sac/Ocn: 0.82 vs. 0.9 x100µm^-1s^-1, p=n.s.). Importantly, Ocn alone did not significantly change CaSpF under basal conditions (Con vs. Ocn 0.33 vs. 0.24 x100µm^-1s^-1, p=n.s.) as well as under stimulation with ATX-II (ATX-II vs. ATX-II+Ocn: 0.53 vs. 0.46 x100µm^-1s^-1, p=n.s.). In line with these results, NPR-C agonist cANP_4-23 significantly reduced CaSpF compared to sole ATX-II treatment (ATX-II vs. ATX-II+cANP_4-23: 0.69 vs. 0.23 x 100µm^-1s^-1, p=0.006). Both, CNP and Sac, did not significantly alter systolic calcium transients compared to ATX-II (each p= n.s.). SR-Ca^2+-load stayed unchanged upon stimulation with CNP and Sac. Preliminary data shows that Sac did not influence cellular cGMP levels, which may exclude a GC-B-dependent- and hint towards a NPR-C-dependent mechanism.

Conclusion
Sacubitrilat exerts significant direct antiarrhythmic effects by reducing diastolic SR- calcium leak without a negative impact on systolic calcium transients and SR-Ca²⁺-load. These effects appear to be mediated through an NPR-C dependent mechanism. The findings support further investigation of natriuretic peptide signaling as a target for antiarrhythmic therapies in HF patients.