CASK is required for the efficacy of CaMKII-inhibitors AIP and AS105

Background:
Ca/Calmodulin-dependent Kinase II (CaMKII) is a key enzyme for the development of heart failure and arrhythmias. We have previously shown that the protein Calcium-Serine-Kinase (CASK) acts as a negative regulator of CaMKII and mediates the inhibitory effects of GLP1-agonists (e.g. Liraglutide) on CaMKII. The exact mechanism of CaMKII-inhibition via CASK remains unclear.

Methods:
To investigate the importance of CASK for CaMKII inhibition, Wildtype (WT)- and CASK-Knock-out (CASK KO)-mice were compared in each experiment. We investigated both the substrate-competitive CaMKII-inhibitor Autocamtide-2-related inhibitory peptide (AIP, 2µM) as well as the ATP-competitive CaMKII-inhibitor AS105 (2µM) to exclude differences relating to the mode of CaMKII inhibition. After preincubation of isolated ventricular cardiomyocytes with either inhibitor, CaMKII was activated with H₂O₂-induced reactive oxygen species (100µM, 5 min). Under these conditions, we investigated the developed force of murine ventricular muscle strips as well as the CaMKII-dependent and proarrhythmogenic late sodium current (late I_Na) in isolated ventricular cardiomyocytes via the patch-clamp-technique. Moreover, we investigated the effect of AIP on systolic Ca transients via epifluorescence microscopy in isolated cardiomyocytes.

Results:
Contractile force of WT muscle strips was reduced upon H₂O₂-influence compared to the vehicle control group (normalized to vehicle, Veh: n=23, 1.0 ± 0.0 vs. H₂O₂: n=23, 0.825 ± 0.032). In WT, AIP and AS105 restored contractile force upon H₂O₂ (H₂O₂ + AIP: n=5, 1.046 ± 0.032 vs. H₂O₂ + AS105: n=5, 1.623 ± 0.094). In contrast to WT muscle strips, neither AIP nor AS105 were able to ameliorate the reduced contractile force in muscle strips from CASK KO-mice. Moreover, the patch-clamp-experiments demonstrated that the pathologically increased late I_Na upon H₂O₂ was reduced by AIP and AS105 in WT (H₂O₂: n=5, -74.29 ± 6.943 vs. H₂O₂ + AIP: n=5, -29.48 ± 5.608 vs. H₂O₂ + AS105: n=2, -36.67 ± 13.97), but not in cardiomyocytes from CASK KO-mice. Furthermore, late I_Na was increased in CASK KO at baseline, but H₂O₂ was, interestingly, not able to further elevate late I_Na (CASK KO: Veh: n=5, -57.91 ± 6.737 vs. H₂O₂: n=7, -60.80 ± 3.099). In WT, AIP was able to ameliorate reduced Calcium transient amplitudes upon H₂O₂ (H₂O₂: n=7, 0.401 ± 0.039 vs. H₂O₂ + AIP: n=7, 0.564 ± 0.074). However, AIP showed no efficacy on Calcium transients upon H₂O₂ in CASK KO.

Discussion:
Our data shows that the effects of specific CaMKII-inhibition with AIP and AS105 on the developed contraction force of cardiomyocytes, late I_Na and Ca transients upon H₂O₂ depend on the presence of the CaMKII inhibitory protein CASK. This suggests that CASK plays a central role in CaMKII-inhibition and its mechanism needs to be further elicited for the understanding of cardiovascular drugs.