Leucine-rich repeat-containing protein 10 (LRRC10) is a cardiac-specific potentiator of the L-type calcium channel/current (LTCC/I Ca,L ) previously studied in heterologous expression systems and animal models. However, its functional importance in the context of human cardiac biology remains unknown. To better understand the roles of LRRC10 in the modulation of LTCC and calcium cycling in human cardiomyocytes, LRRC10 −/− human induced-pluripotent stem cells were generated from the wild-type (WT) DF19-9-11T line using CRISPR-Cas9 gene editing and differentiated to cardiomyocytes (hiPSC-CMs). Whole-cell voltage-clamp experiments demonstrated that LRRC10 −/− hiPSC-CMs exhibited a markedly reduced magnitude of I Ca,L compared to WT ( LRRC10 −/− −15.01 ± 1.77 pA/pF, WT −29.40 ± 2.24 pA/pF; p < 0.05). Nevertheless, action potential duration at 90% repolarization (APD 90 ) in LRRC10 −/− hiPSC-CMs (162.80 ± 13.16 ms) was not significantly different from WT (156.60 ± 10.67 ms). Optical mapping of calcium transients (CaTs) from hiPSC-CM monolayers showed comparable CaT rise time between LRRC10 −/− and WT hiPSC-CMs. To test whether phosphorylation of LRRC10 affects its LTCC modulatory function, we substituted four phosphorylation sites on LRRC10 (T4, Y20, T244, and Y255) with either alanine or glutamate to generate quadruple phosphonull or phosphomimetic LRRC10 mutants (LRRC10-4A or LRRC10-4E, respectively). Re-expression of wild-type LRRC10 by transient transfection in LRRC10 -/- hiPSC-CMs restored I Ca,L magnitude (LRRC10-WT −31.45 ± 2.28 pA/pF, GFP (transfection control) −20.15 ± 1.69 pA/pF; p < 0.05). LRRC10 -/- hiPSC-CMs transfected with either LRRC10-4A or LRRC10-4E had comparable I Ca,L (LRRC10-4A −28.37 ± 2.52 pA/pF, LRRC10-4E −30.81 ± 2.32 pA/pF; NS), which were also not significantly different from LRRC10-WT-transfected hiPSC-CMs. In conclusion, LRRC10 increases I Ca,L in hiPSC-CMs independently of its phosphorylation status, but homeostatic compensation maintains comparable APD and CaT.
Siri-Angkul et al. (Sun,) studied this question.