CRISPR-Cas base editing to eliminate CaMKIIδ autophosphorylation as a therapy against arrhythmias

Introduction

Cardiovascular disease is the main cause of mortality worldwide, with a considerable proportion attributable to arrhythmias. The cardiac stress-responsive enzyme CaMKIIδ promotes arrhythmias by increasing the sarcoplasmic reticulum (SR) Ca2+ leak and late Na+ current. CaMKIIδ undergoes hyperactivation by autophosphorylation at threonine-287. We previously developed a CRISPR-Cas adenine base editing strategy to render CaMKIIδ phospho-resistant and generated a knock-in mouse model (T287A).

Aims

To evaluate the antiarrhythmic potential of inhibiting CaMKIIδ autophosphorylation and develop a postnatal in vivo strategy to render CaMKIIδ phospho-resistant.

Methods

Ventricular cardiomyocytes were isolated from wildtype (WT) and autophosphorylation-resistant CaMKIIδ-T287A knock-in mice and treated with 100 nM isoprenaline (ISO). SR Ca2+ leak was quantified by confocal microscopy. Action potentials and late Na+ current were measured using patch clamp technique. Ca2+ transients were analysed by epifluorescence microscopy. Our previously developed CRISPR-Cas adenine base editing strategy was packaged into an adeno-associated virus with an increased tropism for cardiomyocytes (MyoAAV4A). Ventricular arrhythmias were studied in WT mice by right heart catheterization and electrical burst stimulation. The severity of arrhythmias was scored from 0 (none) to 3 (sustained >10 s) and averaged over five bursts.

Results

In WT but not in T287A cardiomyocytes, ISO increased the Ca2+ spark frequency resulting in a higher proarrhythmogenic diastolic SR Ca2+ leak (Fig. 1A). In WT cardiomyocytes upon ISO, we found a substantial prolongation of action potential duration at 80% repolarization from 30.1 ± 2.7 ms to 61.7 ± 4.3 ms (p<0.001), while T287A cardiomyocytes were protected (44.4 ± 5.0 ms upon ISO, p=0.006 vs. WT+ISO). Accordingly, ISO enhanced the pro-arrhythmic late Na+ current in WT but not in T287A cardiomyocytes (Fig. 1B).

Ca2+ transient amplitudes were increased by ISO in both WT (from [ΔF340/F380] 0.17 ± 0.02 to 0.34 ± 0.04, p=0.002) and T287A (from 0.19 ± 0.02 to 0.35 ± 0.03, p<0.001) cardiomyocytes. Notably, ISO led to an 11-fold increase of spontaneous, arrhythmogenic Ca2+ transients in WT but not in T287A cardiomyocytes (Fig. 1C).

To increase the translational impact, we next subjected adult WT mice to postnatal CRISPR-Cas gene editing. Consistent with our cellular findings, edited mice showed a substantial and significant reduction in arrhythmia severity compared to mice treated with a non-editing control AAV (Fig. 1D).

Conclusion

Cardiomyocytes with a phospho-resistant CaMKIIδ are protected from an increased diastolic SR Ca2+ leak, prolonged action potentials, enhanced late Na+ current, and pro-arrhythmic spontaneous Ca2+ transients. This translates into a reduced risk of ventricular arrhythmias in vivo in adult mice following postnatal gene editing of CaMKIIδ, which highlights its therapeutic potential.Figure 1Figure 2