Pediatric Dilated Cardiomyopathy‐Associated LRRC10 (Leucine‐Rich Repeat–Containing 10) Variant Reveals LRRC10 as an Auxiliary Subunit of Cardiac L‐Type Ca ²⁺ Channels

Figures

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  Figure 1. Identification of a homozygous recessive LRRC10 (leucine‐rich repeat–containing 10) variant in sporadic pediatric dilated cardiomyopathy. A, Family pedigree. Square=male; circle=female; solid=affected; open=unaffected. B, Trio‐based filtering scheme for whole‐exome sequencing data. The number of variants identified in the patient are shown. C, Sanger sequencing verification of the I195T variant in the family quartet. D, Gene structure of LRRC10. LRRC10 consists of a single coding exon and contains 7 leucine‐rich repeats, characterized by a highly conserved segment (LxxLxLxxNxL, where L indicates an isoleucine, a leucine, or a valine residue) and a variable segment. Red indicates the location of the I195T variant, within the seventh leucine‐rich repeat, at the second conserved “L” position. 1000G indicates 1000 Genomes; DCM, dilated cardiomyopathy; EVS, Exome Variant Server; ExAC, Exome Aggregation Consortium; HCS, highly conserved segment; LRR, leucine‐rich repeat; MAF, minor allele frequency.

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  Figure 2. Impact of wild‐type (WT) LRRC10 (leucine‐rich repeat–containing 10) and the dilated cardiomyopathy–linked I195T variant on ICa,L in HEK293 cells. Functional effects of coexpressing WT or I195T LRRC10 with the Cav1.2 complex. A, Representative ICa,L traces recorded at −60, −30, 0, and +30 mV from human embryonic kidney (HEK293) cells transiently expressing either the Cav1.2+β2CN2+α2δ channel complex alone or with WT or I195T LRRC10 using the voltage protocol shown in the inset. Mean current–voltage (I‐V) relationship plotted as mean±SEM. B, Voltage dependence of activation (G/Gmax) and (C) steady‐state inactivation (I/Imax) plotted against voltage (mV) were fitted by Boltzmann distributions, as shown by the solid lines. Data are mean±SEM. *P<0.05.

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  Figure 3. LRRC10 (leucine‐rich repeat–containing 10) coexpression does not affect the expression of L‐type Ca2+ channels on the plasma membrane. The surface membrane proteins of HEK293 cells transiently expressing the Cav1.2 complex alone (HA‐Cav1.2+β2CN2+α2δ) or with Myc‐tagged LRRC10 (wild‐type [WT] or I195T) were biotinylated, purified with streptavidin sepharose and immunoblotted (IB) with anti‐HA antibody to detect the HA‐Cav1.2 protein. Representative immunoblot (n=4) demonstrated no change in surface membrane expression of Cav1.2 complex. Starting material (SM) and cell surface (CS) signals were quantified and normalized to HEK293 cells expressing the Cav1.2 complex alone. Data are mean±SEM with P=0.538. HA indicates hemagglutinin.

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  Figure 4. Wild‐type (WT) LRRC10 (leucine‐rich repeat–containing 10) and dilated cardiomyopathy–associated LRRC10–I195T associate with and differentially modulate the Cav1.2 subunit in HEK293 cells. A, Representative immunoblot (n=3) from coimmunoprecipitation experiments of HEK293 cells transiently expressing HA‐Cav1.2 with either Myc‐WT or Myc‐I195T LRRC10 plasmids. Plasmids encoding for the auxiliary β2CN2 or α2δ subunits were not cotransfected. Lysates were immunoprecipitated (IP) with anti‐HA, anti‐Myc, or IgG control antibodies and immunoblotted (IB) with either anti‐HA or anti‐Myc to detect the Cav1.2 or LRRC10 (WT or I195T) proteins, respectively. B, Representative ICa,L traces recorded at −60, −20, and +10 mV from transiently transfected HEK293 cells expressing Cav1.2 alone or with WT LRRC10 showed larger ICa,L compared with cells expressing Cav1.2 alone generated from the voltage protocol shown in the inset. C, Mean current–voltage (I‐V) relationship plotted as mean±SEM, *P<0.05. D, Voltage dependence of activation (G/Gmax) and (E) representative steady‐state inactivation current traces at prepulses −60 and 0 mV with (F) steady‐state inactivation (I/Imax) plotted against voltage…

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  Figure 5. Lrrc10 −/− ventricular myocytes have reduced ICa,L and shifted steady‐state inactivation of ICa,L. A, ICa,L recordings from wild‐type (WT) or Lrrc10 −/− ventricular myocytes at −40, 0, 20, and +40 mV using the voltage protocol in the inset. Mean current–voltage (I‐V) relationship are plotted as mean±SEM *P<0.05. Inset shows mean peak ICa,L at 0 mV separated by sex with sample size shown in bars for 3 male WT mice, 6 female WT mice, 6 for Lrrc10 −/− male mice, and 4 Lrrc10 −/− female mice. B, Representative steady‐state inactivation traces at prepulses −60, −20, 0, and +20 mV. Mean V1/2 of inactivation was shifted to more depolarized potentials in Lrrc10 −/− myocytes compared with WT with data fitted by Boltzmann distributions.

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  Figure 6. Cav1.2 L‐type Ca2+ channel protein is unchanged in Lrrc10 −/− mice and associates with LRRC10 in wild‐type (WT) mouse hearts. A, Lysates were prepared from WT and Lrrc10 −/− mouse hearts (n=4 each group), and 100 µg of input protein was loaded. Representative immunoblot shows expression of Cav1.2 channel protein and GAPDH loading control in WT and Lrrc10 −/− lysates. Only LRRC10 (leucine‐rich repeat–containing 10) protein is expressed in WT, not Lrrc10 −/− lysates. Normalized Cav1.2 protein signal (Cav1.2/GAPDH) was analyzed by Student t test and found not significantly different between WT and Lrrc10 −/−. B, Representative immunoblot of WT and Lrrc10 −/− mouse heart lysates immunoprecipitated with anti‐LRRC10 or anti‐IgG control antibodies and immunoblotted with anti‐Cav1.2 or anti‐LRRC10 antibodies with the arrows indicating Cav1.2 and LRRC10, respectively. The immunoblot is representative of 3 independent experiments. *IgG heavy and light chains, which are ≈52 and 30 kDa, respectively.