The novel SCN5A-P1891A mutation is associated with left ventricular hypertrabeculation and links Nav1.5 to cardiomyocyte proliferation and disrupted 3D cardiac tissue formation
BackgroundLeft ventricular hypertrabeculation (LVHT) is a heterogenous cardiac condition with a complex and poorly understood aetiology. We comprehensively characterised the effect of a novel P1891A mutation in the SCN5A gene, which encodes the voltage-gated sodium channel Nav1.5, identified in a Finnish family diagnosed with LVHT. MethodsWe generated SCN5A-P1891A mutation-carrying human induced pluripotent stem cell-derived cardiomyocytes (P1891A-hiPSC-CMs) and performed electrophysiological assessments, including patch-clamp studies, and fluorescent calcium imaging, to determine the mutations effect on hiPSC-CM electrophysiology. We also evaluated the impact of the mutation on the proliferative capacity in response to mitogenic stimuli and on the hypertrophic response following cyclic mechanical stretch or endothelin-1 treatment. Further, we assessed the effect on contractile parameters in three-dimensional (3D) contractile hydrogels (engineered heart tissues, EHTs) and conducted advanced proteomics to understand the consequences of the mutation on Nav1.5 protein-protein interactions. ResultsThe SCN5A-P1891A mutation reduced the sodium current densities and increased both the sodium window current and arrhythmogenicity; however, action potential parameters were unaffected. Advanced proteomics characterised, for the first time, the complete Nav1.5 interactome and revealed that the SCN5A-P1891A mutation negated interactions with fibroblast growth factor 12 (FGF12) and FGF13, that are known to modulate sodium channel activity. Baseline proliferation was unchanged, although aged P1891A-hiPSC-CMs demonstrated enhanced proliferative capacity following mitogenic stimulation. Further, P1891A-hiPSC-CMs exhibited a heightened stress response upon mechanical stretch, resulting in the upregulation of heart failure-associated genes. Strikingly, EHTs derived from P1891A-hiPSC-CMs yielded disparate phenotypes. Whilst the majority condensed only partially and failed to beat synchronously, a small subset condensed fully yet exhibited weak contractile properties, alongside age-associated functional decline. In contrast, EHTs derived from healthy control hiPSC-CMs consistently condensed fully and demonstrated a positive correlation between post-fabrication age and contractile properties ConclusionsOur study presents a unique aetiology of LVHT and reveals a novel association between SCN5A mutations and enhanced human cardiomyocyte proliferation. Further, the inability of P1891A-hiPSC-CMs to consistently form fully condensed 3D cardiac tissues may be linked to their abnormal response to mechanical stretch and provides a powerful 3D model for future mechanistic research and drug development studies to better understand and treat LVHT.