Introduction:
Arrhythmogenic cardiomyopathy (ACM) is a hereditary cardiac disease characterized by cardiomyocyte loss and progressive fibro-fatty replacement associated with arrhythmias, heart failure or sudden cardiac death. ACM is mainly caused by desmosomal mutations; the most common disease-associated gene is Plakophilin-2 (PKP2). Mutant desmosomal proteins result in disturbed desmosomal integrity and thereby loss of proper cell-cell adhesion. More recently, inflammatory and autoimmune processes have been attributed to be involved in the pathogenesis of the disease.

Purpose:
To gain insights in the pathomechanisms of ACM and involved inflammatory signaling using PKP2 deficient (PKP2-KO) and isogenic control induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).

Methods and results:
PKP2-KO gene-edited hiPSCs and their isogenic control hiPSC line were used after in vitro differentiation into hiPSC-CMs and 60 days of maturation. Pro-inflammatory agents like lipopolysaccharide (LPS), interleukin-1β (IL-1β) or tumor necrosis factor α (TNFα) were added and should activate inflammatory signaling pathways such as p38 mitogen activated protein kinase (MAPK) and nuclear factor κB (NFκB) signaling and thereby decrease cellular adhesion. Immunofluorescence staining of Neuronal-Cadherin (N-Cadherin) and quantification of cell-cell contact distances was measured as a readout representing the junctional width of cardiac adherence junctions. After IL-1β (increase of N-Cadherin distance of 19 and 25 % in control and PKP2-KO hiPSC-CMs, respectively) and TNFα stimulation (N-Cadherin distance incline of 14 and 23 % in control and PKP2-KO hiPSC-CMs, respectively), the strongest response on cellular adhesion was observed in both cell lines. LPS had similar impact on control and PKP2-KO hiPSC-CMs. To rescue reduced cellular adhesion after pro-inflammatory treatment, SB202190 and BAY11_7082, inhibitors of p38 MAPK and NFκB signaling were used. PKP2-KO hiPSC-CMs showed a higher reaction towards p38 MAPK inhibition mediated by SB202190, indicating a potentially increased p38 MAPK signaling in PKP2-KO hiPSC-CMs. NFκB inhibition rescued pro-inflammatory increase of N-Cadherin distance in both cell lines to a similar level, indicating possibly similar NFκB activity. Additionally, a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) was used to directly disturb cellular connectivity by DSG2 or N-Cadherin cleavage. A higher impact of ADAM10 was present in PKP2-KO hiPSC-CMs (19 % increase of N-Cadherin distance in PKP2-KO vs. 5 % in control hiPSC-CMs). SB202190, BAY11_7082 and the ADAM10 inhibitor GI254023X rescued ADAM10 dependent increase of N-Cadherin distance.

Conclusion:
In summary, the pro-inflammatory agents LPS, TNFα, IL-1β and ADAM10 increased N-Cadherin distance to different levels. In PKP2-KO hiPSC-CMs, ADAM10 had higher impact while control hiPSC-CMs showed no significant response indicating specificity to this pathway. Rescue of the increase of N-Cadherin distance after pro-inflammatory treatment was successful using p38 MAPK, NFκB or ADAM10 inhibitors. These results support that hiPSC-CMs enable in vitro modeling of pathogenic inflammatory signaling pathways that are involved in ACM. Currently, cell-cell adhesion via dissociation assays is investigated to deliver insights on functional consequences of healthy and ACM-mutant hiPSC-CMs to inflammatory stimuli.