Abstract Body: Background: Leucine-rich repeat-containing protein 10 (LRRC10) is a novel regulator of the cardiac L-type Ca²⁺ channel/current (LTCC/I_Ca,L). LRRC10 is crucial for cardiac regeneration in multiple species, and LRRC10 variants have been associated with dilated cardiomyopathy in humans. LRRC10 increases I_Ca,L in HEK-293 cells expressing rabbit Ca_V1.2 subunit, and knockout of LRRC10 in zebrafish and mouse cardiomyocytes (CM) reduces I_Ca,L. However, LRRC10-mediated regulation of human cardiac LTCC has not been demonstrated, and the mechanism by which LRRC10 modulates LTCC is not known. Moreover, the effect of LRRC10 on the T-type Ca²⁺ channel/current (TTCC/I_Ca,T), which has been implicated in CM cell cycle regulation, has never been tested.
Methods: LRRC10 knockout (LRRC10^–/–) human induced-pluripotent stem cells (hiPSC) were generated from wild-type (WT) DF19-9-11T hiPSCs using CRISPR-Cas9 techniques. LRRC10^–/– and WT hiPSCs were differentiated to CMs using the standard GiWi protocol. Whole-cell I_Ca,L and I_Ca,T were examined in LRRC10^–/– and WT hiPSC-CMs. Whole-cell I_Ca,L was also assessed in HEK-293 cells expressing human cardiac LTCC subunits (Ca_V1.2, β₂, and α₂δ) with or without human LRRC10. To identify the LRRC10-interacting site of Ca_V1.2, GST-pulldown assays were performed using purified GST-fusion constructs of the intracellular domains of rabbit Ca_V1.2 and lysates from HEK-293 cells expressing human LRRC10. Ca_V protein sequences were analyzed using the Clustal Omega program.
Results: Genetic ablation of LRRC10 in hiPSC-CMs decreased peak I_Ca,L (WT -29.4±2.2 pA/pF, LRRC10^–/– -15.0±1.8 pA/pF, p<0.01) but did not significantly affect I_Ca,T magnitude (WT -3.9±0.5 pA/pF, LRRC10^–/– -4.0±0.7 pA/pF, p>0.05 [NS]). LRRC10 increased peak I_Ca,L in HEK-293 cells expressing human LTCC (with LRRC10 -156.0±19.8 pA/pF, without LRRC10 -60.2±12.0 pA/pF, p<0.05). LRRC10 was pulled down by the GST-Ca_V1.2-N-terminus but not by other GST-Ca_V1.2 constructs. Sequence analyses revealed an invariant exon 2-encoded segment of the N-terminus (NT) that was present in both human and rabbit LTCC Ca_V1.2 but not in TTCC Ca_V3.1 and Ca_V3.2.
Conclusions: The Ca²⁺ influx-potentiating effect of LRRC10 is conserved in human LTCC and is specific to regulate I_Ca,L, not I_Ca,T. The invariant Ca_V1.2 NT segment is a potential LRRC10-interacting site. The LRRC10^–/– hiPSC line is a promising tool for future investigations into the roles of LRRC10-LTCC interaction in human cardiac biology and disease.