Decreased Contraction Rate, Altered Calcium Transients, and Increased Proliferation seen in Patient‐specific iPSC‐CMs Modeling Ebstein’s Anomaly and Left Ventricular Noncompaction

Patients presenting with Ebstein’s anomaly (EA) have right heart dysfunction, mainly tricuspid valve insufficiency, and often manifest with left ventricular noncompaction (LVNC). Apart from a few sarcomeric variant associations, the genetic etiology and pathogenesis of combined EA/LVNC remain largely unknown. In a familial case where 10 of 17 members, including four fetal deaths, inherited EA/LVNC in an autosomal dominant pattern, we discovered a novel and damaging missense variant in the gene KLHL26 that segregates with disease. The KLHL26 (p.R237C) variant comprises an altered electrostatic surface profile, likely decoupling the CUL3‐interactome and altering protein turnover. Objective Our in vitro patient‐specific studies enable the discovery of EA/LVNC developmental pathways and possibly related forms of heart failure and cardiomyopathy that may inform early clinical interventions and improve long‐term patient outcomes. Hypothesis We hypothesize that the KLHL26 variant is etiologic for this familial EA/LVNC case. Methods We differentiated a family trio (a heart‐healthy daughter and EA/LVNC‐affected mother and daughter) of induced pluripotent stem cells into cardiomyocytes (iPSC‐CMs) in a blinded manner on three iPSC clones per subject. Using flow cytometry, immunofluorescence, and biomechanical, electrophysiological, and automated contraction methods, we investigated iPSC‐CM differentiation efficiency between D10‐20, contractile properties and cell cycle regulation at D20, and sarcomere organization at D60. We further conducted differential analyses following label‐free protein and RNA‐Seq quantification at D20. Via CRISPR‐Cas9 gene editing, we plan to characterize KLHL26 variant‐specific iPSC‐CM alterations and connect findings to discoveries from patient‐specific studies. Results All iPSC lines differentiated into CMs with an increased percentage of cTnT+ cells in the affected daughter line. In comparison to the unaffected, affected iPSC‐CMs had fewer contractions per minute and altered calcium transients, mainly a higher amount of total calcium release, faster rate of rise and faster rate of fall. The affected daughter line further had shorter shortening and relaxation times, higher proliferation, lower apoptosis, and a smaller cell surface area per nucleus. The affected mother line trended in a similar direction to the affected daughter line. There were no gross differences in sarcomere organization between the lines. We also discovered differential expression of candidate proteins such as kinase VRK1 and collagen COL5A1 from proteomic profiling. Discussion These discoveries suggest that EA/LVNC characteristics or pathogenesis may result from decreased contractile ability, altered calcium transients, and cell cycle dysregulation. Through CRISPR‐Cas9 correction and introduction of the KLHL26 variant in the daughter lines, we will build upon this understanding to inform exploration of critical clinical targets for EA/LVNC and related forms of cardiomyopathy and heart failure.