Cardiac Electrophysiological Anomalies Associated with A Noonan Syndrome Mutation in RAF1 Can Be Rescued Partially In Vitro by Inhibition of RAS/MAPK Signaling Pathway

Objectives: Noonan syndrome (NS) is known to be highly associated with hypertrophic cardiomyopathy (HCM) and affected patients are at particular risk of developing arrhythmias. Individuals bearing the Noonan syndrome-associated gain-of-function mutation c.770C>T in RAF1 resulting in RAF1S257L/+ show severely dysfunctional myocardium.

Methods: To elucidate the role of RAF1S257L/+ on cardiac (electro-) physiology, we used highly purified cardiomyocytes (CMs) differentiated from patient-derived induced pluripotent stem cells (iPSCs) to generate rod-shaped 3D bioartificial cardiac tissues (BCTs) in vitro. Contractile force was analyzed by a multimodal bioreactor system and electrophysiological characteristics were evaluated by sharp micro-electrode and multi electrode array (MEA) approaches.

Result: In comparison to wild type (WT) BCTs, we showed that RAF1S257L/+ BCTs phenocopied the clinical myocardial pathological characteristics by exhibiting a substantial tissue enlargement (1.1 vs. 0.7 mm², p < 0.0001), indicating HCM, combined with impaired contractile tension (3.9 vs. 7.1 kPa, p < 0.0001). Electrophysiological analysis revealed a ≈40% higher spontaneous beating frequency (1.1 Hz vs. 0.7 Hz, p < 0.0001) and significantly shorter field potential duration (FPD) in RAF1S257L/+ BCTs versus WT BCTs (243 vs. 316 milliseconds, p < 0.0001). In addition, inhibition of MEK activity was explored to prevent the observed alterations in RAF1S257L/+ BCT functionality. Long-term treatment with MEK inhibitor (MEKi) significantly reduced cross-sectional area (0.9 ± 0.1 mm2) while enhancing contractile tension of the RAF1S257L/+ BCTs (4.5 ± 0.4 kPa), suggesting the partial phenotype rescue. Furthermore, MEKi treated RAF1S257L/+ BCTs exhibited a reduced spontaneous beating frequency (0.9 vs. 1.1 Hz, p < 0.05) and ≈30% longer FPDc. Accordingly, sharp electrode measurements confirmed significantly longer action potentials compared with non-treated RAF1S257L/+ BCTs (425 vs. 296 milliseconds, p < 0.0001). Additionally, roaming pacemaker regions with unstable excitation patterns, resulting in spontaneous ectopic foci in RAF1S257L/+ BCTs, were stabilized by treatment with MEKi.

Conclusion: Our study provides an in-depth (electro-) physiological characterization of RAF1S257L/+-associated cardiac manifestations related to NS, i.e., myocardial thickening, impaired contractility and altered electrophysiological properties. Moreover, we were able to partially rescue the phenotype by pharmacological intervention.

Publication History

Article published online:
19 February 2021