Acute activation of protein phosphatase-1 by a novel photoactivatable protein phosphatase-1 disrupting peptide prevents cellular arrhythmias in murine cardiomyocytes

The development of an increased diastolic SR calcium leak due to hyperphosphorylation of ryanodine receptor 2 (RyR2) constitutes an important pathomechanism in heart failure and atrial fibrillation, impairing myocardial contractility and promoting cellular arrhythmias. In previous studies, we could significantly reduce SR calcium sparks (CaSp) and arrhythmogenic SR calcium waves (CaWa) in a murine arrhythmia model as well as human terminal heart failure by activating protein phosphatase-1 (PP1) through a novel PP1 disrupting peptide (PDP), without compromising SR calcium load or systolic calcium transients (Fischer et al., Eur J Heart Fail 2018; Eiringhaus et al., Basic Res Cardiol 2019). However, the required incubation time of the investigated peptide PDP3 complicates the differentiation between acute effects of PP1 activation on EC coupling and metabolic effects with longer latency.
In our current study, we therefore used the novel compound PDPcaged, a photoactivatable peptide based on PDP-Nal, a PDP exhibiting higher in vitro potency and light stability than PDP3. In murine ventricular Cardiomyocytes (CM) stimulated with Isoprenaline (Iso, 30 nM), PDP-Nal showed the expected reduction of CaSp frequency in comparison to the inactive control peptide PDPm-Nal (0,91 vs. 1,32 100 µm^-1*s^-1; n= 75 vs. 79; P< 0,05). To investigate the acute effects of PP1-activation on SR calcium cycling we pre-incubated murine CM with PDPcaged and Iso (30 nM). In the intervention group (PDPcage UV⁺), PDPcaged was activated by UV-illumination (LED 365 nm, 5 min.) immediately before start of measurements. Again, our preliminary data show a tendency towards a reduced CaSp frequency (0,94 vs. 1,50 100 µm^-1*s^-1; n= 34 vs. 24; P= 0,14; Fig. 1B) after UV-induced activation of PDPcaged in comparison to the non-UV-illuminated CM (PDPcage UV^-). Furthermore, we could detect a significantly reduced frequency of arrhythmogenic CaWa (0,26 vs. 0,60 100 µm^-1*s^-1; n= 35 vs. 27, P< 0,01; Fig. 1C) in the intervention group. The control group (Iso UV⁺: UV-illuminated CM without PDPcaged) showed neither an increased CaSp nor CaWa frequency as a possible indication of phototoxicity.


Fig. 1: A) Representative confcocal line-scans (Fluo4-AM 10 µM, Iso 30 nM) of isolated murine ventricular CM. Analysis of B) CaSp frequency and C) CaWa frequency.

In summary, these findings confirm an acute activation of PP1 as a promising therapeutic strategy targeting arrhythmias and contractile dysfunction in heart failure and atrial fibrillation. The results obtained using the novel photoactivatable peptide PDPcaged suggest for the first time that these effects can be primarily attributed to an acute modification of cellular calcium cycling and not to subacute effects on cardiac metabolism or protein biosynthesis. We will further elaborate these results by analysing SR calcium load and systolic calcium release with epifluorescence microscopy and phosphorylation status of key calcium handling proteins by Western Blot experiments.