Abstract 503 Pro-inflammatory cytokines affect cell-cell contacts in healthy and plakophilin-2 deficient hiPSC-cardiomyocytes

Introduction:

Arrhythmogenic cardiomyopathy (ACM) is a hereditary cardiac disease associated with cardiomyocyte loss, progressive fibro-fatty replacement as well as inflammation of myocardial tissue. ACM is caused by mutations in cardiac cellular junctions like desmosomes with plakophilin-2 (PKP2) as the most common affected protein. Mutant desmosomal proteins disturb desmosomal integrity and cause a loss of cellular adhesion. Presumably, inflammatory and autoimmune processes are connected to the ACM phenotype either as a trigger for disease progression or as a direct effect on desmosome structure and signaling.

Purpose:

To gain insights on the effects of pro-inflammatory stimulation on cell-cell contacts, adhesion and calcium homeostasis of PKP2 deficient (PKP2-KO) and control induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to further decipher the pathomechanism of ACM.
Methods and Results:

Isogenic control (Ctr) and gene-edited PKP2-KO hiPSCs were differentiated into hiPSC-CMs and maturated for 60 days. Under the hypothesis that pro-inflammatory stimulation with interleukin-1β (IL-1β) or tumor necrosis factor α (TNFα) activates inflammatory signaling pathways like p38 mitogen activated protein kinase (MAPK) or nuclear factor κB (NFκB) and thereby decreases cellular adhesion; Ctr and PKP2-KO hiPSC-CMs were treated with cytokines for 24 hours and analyzed. First, changes in cell-cell contact distances were obtained from quantification of immunofluorescent staining of Neuronal-Cadherin (N-Cadherin), representing the junctional width of cardiac adherence junctions. Here, we could show an increase of junctional width of up to 25 % in treated PKP2-KO and Ctr hiPSC-CMs compared to the respective untreated controls. Furthermore, rescue experiments by simultaneous application of cytokines and p38 MAPK or NFκB inhibitors resulted in control-like junctional widths, indicating that hiPSC-CMs react to pro- and anti-inflammatory stimulation. Next, we performed dissociation assays as a direct readout of cellular adhesion, which is based on an enzymatic step to detach hiPSC-CMs from the cultivation plate followed by mechanical stress to dissociate the cell layer. We started to analyze the fragment count at baseline, where PKP2-KO hiPSC-CMs showed an increased number of fragments compared to Ctr hiPSC-CMs. Pro-inflammatory stimulation resulted in a pronounced increase of fragment counts in both cell lines, resembling decreased cellular adhesion upon stimulation. Loss of PKP2 not only interferes with cellular adhesion, but also with intracellular calcium homeostasis. First experiments on PKP2-KO compared to Ctr hiPSC-CMs revealed altered Ca²⁺ amplitudes and elevated intracellular systolic [Ca²⁺] in PKP2-KO hiPSC-CMs. Further validation is required as well as results how pro-inflammatory stimulation affects calcium homeostasis in PKP2-KO and Ctr hiPSC-CMs.

Conclusion:

In summary, pro-inflammatory agents such as TNFα and IL-1β increased junctional widths in healthy and PKP2-KO hiPSC-CMs, which was reversible using p38 MAPK and NFκB inhibitors. Dissociation assays showed decreased cellular adhesion in PKP2-KO hiPSC-CMs at baseline and after pro-inflammatory stimulation in both, mutant and Ctr hiPSC-CMs indicating a sufficient model to investigate inflammation and treatment responses on cell adhesion in vitro. Further experiments elucidating changes in calcium homeostasis after pro-inflammatory stimulation will be conducted.

Clin Res Cardiol (2023). https://doi.org/10.1007/s00392-023-02180-w
Pro-inflammatory cytokines affect cell-cell contacts in healthy and plakophilin-2 deficient hiPSC-cardiomyocytes
K. Walz¹, A. Hartleb¹, M. Kohlhaas¹, A. Janz¹, C. Maack¹, B. Gerull¹
¹Deutsches Zentrum für Herzinsuffizienz, Universitätsklinikum Würzburg, Würzburg;
Introduction: Arrhythmogenic cardiomyopathy (ACM) is a hereditary cardiac disease associated with cardiomyocyte loss, progressive fibro-fatty replacement as well as inflammation of myocardial tissue. ACM is caused by mutations in cardiac cellular junctions like desmosomes with plakophilin-2 (PKP2) as the most common affected protein. Mutant desmosomal proteins disturb desmosomal integrity and cause a loss of cellular adhesion. Presumably, inflammatory and autoimmune processes are connected to the ACM phenotype either as a trigger for disease progression or as a direct effect on desmosome structure and signaling. Purpose: To gain insights on the effects of pro-inflammatory stimulation on cell-cell contacts, adhesion and calcium homeostasis of PKP2 deficient (PKP2-KO) and control induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to further decipher the pathomechanism of ACM. Methods and Results: Isogenic control (Ctr) and gene-edited PKP2-KO hiPSCs were differentiated into hiPSC-CMs and maturated for 60 days. Under the hypothesis that pro-inflammatory stimulation with interleukin-1β (IL-1β) or tumor necrosis factor α (TNFα) activates inflammatory signaling pathways like p38 mitogen activated protein kinase (MAPK) or nuclear factor κB (NFκB) and thereby decreases cellular adhesion; Ctr and PKP2-KO hiPSC-CMs were treated with cytokines for 24 hours and analyzed. First, changes in cell-cell contact distances were obtained from quantification of immunofluorescent staining of Neuronal-Cadherin (N-Cadherin), representing the junctional width of cardiac adherence junctions. Here, we could show an increase of junctional width of up to 25 % in treated PKP2-KO and Ctr hiPSC-CMs compared to the respective untreated controls. Furthermore, rescue experiments by simultaneous application of cytokines and p38 MAPK or NFκB inhibitors resulted in control-like junctional widths, indicating that hiPSC-CMs react to pro- and anti-inflammatory stimulation. Next, we performed dissociation assays as a direct readout of cellular adhesion, which is based on an enzymatic step to detach hiPSC-CMs from the cultivation plate followed by mechanical stress to dissociate the cell layer. We started to analyze the fragment count at baseline, where PKP2-KO hiPSC-CMs showed an increased number of fragments compared to Ctr hiPSC-CMs. Pro-inflammatory stimulation resulted in a pronounced increase of fragment counts in both cell lines, resembling decreased cellular adhesion upon stimulation. Loss of PKP2 not only interferes with cellular adhesion, but also with intracellular calcium homeostasis. First experiments on PKP2-KO compared to Ctr hiPSC-CMs revealed altered Ca²⁺ amplitudes and elevated intracellular systolic [Ca²⁺] in PKP2-KO hiPSC-CMs. Further validation is required as well as results how pro-inflammatory stimulation affects calcium homeostasis in PKP2-KO and Ctr hiPSC-CMs. Conclusion: In summary, pro-inflammatory agents such as TNFα and IL-1β increased junctional widths in healthy and PKP2-KO hiPSC-CMs, which was reversible using p38 MAPK and NFκB inhibitors. Dissociation assays showed decreased cellular adhesion in PKP2-KO hiPSC-CMs at baseline and after pro-inflammatory stimulation in both, mutant and Ctr hiPSC-CMs indicating a sufficient model to investigate inflammation and treatment responses on cell adhesion in vitro. Further experiments elucidating changes in calcium homeostasis after pro-inflammatory stimulation will be conducted.
https://dgk.org/kongress_programme/jt2023/aP561.html