Loss of LMOD1 impairs smooth muscle cytocontractility and causes megacystis microcolon intestinal hypoperistalsis syndrome in humans and mice

Abstract

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital visceral myopathy characterized by severe dilation of the urinary bladder and defective intestinal motility. The genetic basis of MMIHS has been ascribed to spontaneous and autosomal dominant mutations in actin gamma 2 (ACTG2), a smooth muscle contractile gene. However, evidence suggesting a recessive origin of the disease also exists. Using combined homozygosity mapping and whole exome sequencing, a genetically isolated family was found to carry a premature termination codon in Leiomodin1 (LMOD1), a gene preferentially expressed in vascular and visceral smooth muscle cells. Parents heterozygous for the mutation exhibited no abnormalities, but a child homozygous for the premature termination codon displayed symptoms consistent with MMIHS. We used CRISPR-Cas9 (CRISPR-associated protein) genome editing of Lmod1 to generate a similar premature termination codon. Mice homozygous for the mutation showed loss of LMOD1 protein and pathology consistent with MMIHS, including late gestation expansion of the bladder, hydronephrosis, and rapid demise after parturition. Loss of LMOD1 resulted in a reduction of filamentous actin, elongated cytoskeletal dense bodies, and impaired intestinal smooth muscle contractility. These results define LMOD1 as a disease gene for MMIHS and suggest its role in establishing normal smooth muscle cytoskeletal-contractile coupling.

Keywords: CRISPR-Cas9; Leiomodin; genetics; myopathy; smooth muscle.

Fig. 1.

Loss-of-function mutation in LMOD1 gene of an MMIHS patient. (A) Timed ultrasonography at 28, 30, and 32 wk of gestation showing distended bladder (Bl) and hydronephrotic kidney (K). A postnatal barium enema confirmed MMIHS (Far Right); white arrow and arrowhead indicate the sigmoid colon and descending colon, respectively. (B) In utero ultrasonography of control (a and c) and MMIHS patient (b and d) demonstrating megacystis (Bl) and hydronephrosis (K) in the MMIHS patient. (C) Pedigree of the consanguineal family and results of Sanger sequencing. A heterozygous variant is identified in DNA of the parents and a homozygous variant generating a PTC (TGA) is detected in the proband. (D) RT-qPCR analysis of LMOD1 using RNA from control and patient-derived dermal fibroblasts. (Inset) Protein expression of LMOD1 in control (Ctrl) and patient dermal fibroblasts. Data shown are the mean expression of LMOD1 ± SD of three controls and the patient, performed in triplicate. ***P < 0.001 by unpaired t test.

Fig. 2.

Two-component CRISPR genome editing of mouse Lmod1. (A) Schematic of targeting strategy to disrupt exon one of Lmod1. Injection of Cas9 mRNA and three guide RNAs (red arrowheads) resulted in deletion of 151 bp (red) within exon one of the mouse Lmod1 gene. PCR primers (blue triangles) were used to confirm deletion and sequence changes. (B) PCR products generated with forward and reverse primers (shown in A) indicating WT (+/+), heterozygous (+/−), and homozygous (−/−) deletion of Lmod1. (C) Deletion of the 151-bp region resulted in a frameshift of the ORF and the introduction of a PTC within Lmod1. WT amino acid is in black, and the frame-shifted sequence is in red (asterisk denotes the UGA stop codon). (D) Number of live-born pups of each genotype from heterozygous intercross (P = 0.79 versus expected Mendelian numbers by χ² test). (E) Western blotting of LMOD1 protein in stomach of different Lmod1 genotypes.

Fig. 3.

MMIHS phenotype in Lmod1^−/− mice at embryonic (e) day 18.5. (A) Gross pathology showing distended bladder in Lmod1^−/− mice at e18.5 (white arrow and white bracket) compared with Lmod1^+/+ littermate control. (B) H&E staining shows distended bladder and stomach in the same plane of section from an e18.5 Lmod1^−/− mouse. (Scale bar: 1 mm.) (C) H&E staining and immunofluorescence staining of indicated SMC marker proteins in sections of bladder, intestine, and stomach from e18.5 Lmod1^−/− versus Lmod1^+/+ mice. (Scale bars: 100 μm.)

Fig. 4.

MMIHS phenotype in postnatal Lmod1^−/− mice. (A) Gross image of Lmod1^+/+ (leftmost) and Lmod1^−/− (rightmost) pups showing abdominal distention in the mutant due to bladder and milk-filled stomach (Top). A higher magnified image of a mutant is shown at Bottom. (B) Dissected gross images of stomach and bladder from indicated Lmod1 genotypes. (C) Dissected kidneys and hydronephrosis in some postnatal day 5 Lmod1^−/− mice. (D) H&E-stained sections of kidneys from each genotype. Note the large renal cysts in the Lmod1^−/− section, representing evidence of previously retained water in the mutant kidneys. (E) Sections of Lmod1^+/+ versus Lmod1^−/− stomach and bladder stained with H&E (Top row) or antibodies to the indicated proteins. DAPI nuclear stain was pseudocolored white. (Scale bars: H&E sections, 100 μm; immunofluorescence sections, 50 μm.)

Fig. 5.

Histology and LMOD1 immunofluorescence in three segments of intestine. H&E-stained sections of duodenum, jejunum, and ileum (Left column) and immunofluorescence microscopy of ACTA2 and LMOD1 show absence of LMOD1 protein in visceral smooth muscle of intestinal segments of Lmod1^−/− mice. [Scale bars: H&E sections, 100 μm (vertical line at Top Left); immunofluorescence sections, 50 μm (Bottom Right).] Note, the apparent faint immunostaining for LMOD1 in mutants is nonspecific (SI Appendix, Fig. S4).

Fig. 6.

LMOD1 knockdown in human intestinal SMCs decreases actin filament formation and contractility. (A) In vitro G-actin:F-actin assay in cultured human intestinal smooth muscle cells (hiSMCs) with LMOD1 knockdown. S and P refer to supernatant (G-actin) and pellet (F-actin), respectively. (B) Collagen contractility assays (Left) in hiSMCs transfected with siCtrl or siLMOD1 for 24 or 48 hours (H). Quantitative data are shown at Right with Western blot of LMOD1 protein (Inset). Data shown are mean ± SD of three independent experiments, performed in triplicate. ***P < 0.001 by unpaired t test.

Fig. 7.

Ultrastructure of bladder smooth muscle in postnatal Lmod1^−/− mice. Lmod1^+/+ WT (A and C) versus Lmod1^−/− mutant (B and D) SMC dense bodies (arrowheads) that are much larger in mutants. Boxed regions in A and B are shown at higher magnifications in C and D, respectively. [Scale bars: A and B, 1 μm; C and D, 200 nm (black in Lower Left of each panel).] Arrows point to glycogen granules, which are more prevalent in WT SMCs. (E) Quantitative analysis of dense body length in WT (+/+) versus mutant (−/−) SMCs (n = 35 for WT and n = 37 for mutant). Scatterplot depicts the means ± SD (***P < 0.0001 by paired t test). (F) Quantitative analysis of dense body concentration in WT (+/+) versus mutant (−/−) SMCs (n = 86 for WT and n = 102 for mutant). Scatterplot depicts the mean ± SD (***P < 0.0001 by paired t test from two independent and blinded observers).

Fig. 8.

Loss of LMOD1 results in reduced passive tensile strength and agonist-induced contractility in intestinal rings. (A) Lmod1^−/− jejunum ring segments exhibit decreased tensile strength after dilation using wire myography. Ring segments were mounted and equilibrated before being dilated at set distances, and the passive tension of the ring segment was recorded. (B) Representative myography traces showing force of contraction versus time for control (Ctrl) and mutant (−/−) mouse jejunum ring segments treated with 60 mM KCl or 1 μM carbachol. (C) Quantitation of data from B. Shown are the means ± SD from n = 6 knockout or control groups receiving KCl treatment and n = 4 for carbachol treatment (**P < 0.01 for all graphs by paired t test analysis).