Disruption of cardiac thin filament assembly arising from a mutation in LMOD2: A novel mechanism of neonatal dilated cardiomyopathy

Abstract

Neonatal heart failure is a rare, poorly-understood presentation of familial dilated cardiomyopathy (DCM). Exome sequencing in a neonate with severe DCM revealed a homozygous nonsense variant in leiomodin 2 (LMOD2, p.Trp398*). Leiomodins (Lmods) are actin-binding proteins that regulate actin filament assembly. While disease-causing mutations in smooth (LMOD1) and skeletal (LMOD3) muscle isoforms have been described, the cardiac (LMOD2) isoform has not been previously associated with human disease. Like our patient, Lmod2-null mice have severe early-onset DCM and die before weaning. The infant's explanted heart showed extraordinarily short thin filaments with isolated cardiomyocytes displaying a large reduction in maximum calcium-activated force production. The lack of extracardiac symptoms in Lmod2-null mice, and remarkable morphological and functional similarities between the patient and mouse model informed the decision to pursue cardiac transplantation in the patient. To our knowledge, this is the first report of aberrant cardiac thin filament assembly associated with human cardiomyopathy.

Fig. 1. Representative cardiac imaging and histology from the patient.

(A to C) Echocardiograms from the second day of life, before cannulation onto extracorporeal membrane oxygenation. (A) Apical four-chamber view in systole showing a dilated left atrium, normal size right ventricle (RV), and severely dilated LV without hypertrophy. There are severely diminished RV and LV systolic function and swirling (“smoke”) within the LV cavity. With color Doppler over the mitral valve, there is mild regurgitation. The aortic valve is trileaflet but barely opening, with mild aortic insufficiency in the setting of minimal cardiac output. (B and C) Parasternal long and short axis views, demonstrating severely dilated and globular LV and severely diminished LV and RV systolic function. bpm, beats per minute. (D) Representative electrocardiogram clips of leads II and V5 demonstrating dysrhythmias experienced by the patient before transplant, including monomorphic ventricular tachycardia with a right bundle branch block morphology. (E) Areas of stretched and wavy myocardium are present (hematoxylin and eosin stain, 20×). (F) Foci of somewhat disorganized and hypertrophic myocytes with nuclear crowding are observed; nuclear enlargement and irregularity of nuclear contours as well as perinuclear clearing are quite prominent in certain regions (hematoxylin and eosin, 60×). (G) Marked fibrosis (blue) is present throughout the subendocardium of both ventricles with patchy interstitial nonreplacement-type perimyocyte fibrosis (Masson’s trichrome stain, 10×). (H) Multilayered elastic fibers (black) are also noted in the thickened subendocardium (elastic stain, 10×).

Fig. 2. Lmod2 protein is not detected, mature mRNA levels are reduced, and pre-mRNA levels are largely unchanged in the patient’s LV.

(A) Diagram of the domain layout of human Lmod2 protein with the location of the patient’s nonsense mutation (Trp398*) indicated. Lmod2 contains a tropomyosin binding domain (TM), three actin-binding domains (A1 to A3), and a proline-rich region (P). Actin-binding site 2 is composed of a leucine-rich domain (LRR), while actin-binding site 3 is a Wiskott-Aldrich syndrome protein–homology 2 (WH2) domain (34). (B) Western blots of Lmod2, Tmod1, cardiac troponin I (cTnI), and α-actinin in LV of nonfailing (NF) donor hearts at 48, 24, and 14 months of age and the patient’s (Pt) explanted heart (10 months). Total protein was stained with Ponceau S (PonS); the arrows indicate actin. MW, molecular weight. (C) Relative protein levels following normalization to total protein. Note that the 14-month-old nonfailing donor heart (closest in age to the patient) was set to 1. ND, not detected. Quantitative polymerase chain reaction (qPCR) analysis of Lmod2 mature mRNA (D) and pre-mRNA (E) expression in the LV of a 14-month-old nonfailing donor heart (black bar) or the patient’s explanted heart (gray bar).

Fig. 3. The patient’s heart displays disorganized myofibrils, wide Z-discs, and extraordinarily short thin filaments.

(A) Representative deconvolution immunofluorescence images of LV tissue from a 14-month-old nonfailing donor heart and explanted patient’s heart stained with fluorescently conjugated phalloidin to label F-actin and antibodies to Tmod1 (thin filament pointed end marker) and α-actinin (Z-disc marker). F-actin is in red, and Tmod1 is in green in the merged images. Magenta arrows denote thin filament pointed ends, and Z-discs are marked with a Z. Magenta bars indicate examples of thin filament arrays (one Z-disc with two bundles of thin filaments extending into opposite half-sarcomeres) that were measured. Scale bar, 2.5 μm. (B) Mean LV thin filament lengths measured via Tmod1 staining using the DDecon plugin for ImageJ. Note that the length of one thin filament is one-half of the width of the thin filament array marked in (A). Values are means ± SD; n = 35 and 39 measurements (NF14 and Pt, respectively). ****P < 0.0001, Student’s t test.

Fig. 4. Calcium-activated contractile force is reduced, and the myofilament lattice is desensitized to calcium in cardiomyocytes isolated from the patient’s heart.

The fraction of maximum force produced at various concentrations of free calcium is plotted (sarcomere length, ~1.8 μm). Mean force at maximum calcium activation is indicated in the inset. All values are means ± SEM; n = 7, 8, 8 cells and 4 cell clusters (NF48, NF24, NF14, and Pt, respectively). **P < 0.01 and ***P < 0.001, one-way analysis of variance (ANOVA) with Tukey’s post hoc test.

Fig. 5. Expression of GFP-Lmod2[W405*] reduces cardiac disease progression of Lmod2 knockout mice.

Echocardiography and Western blot analysis of Lmod2 knockout mice injected with adeno-associated virus expressing GFP (black bars), GFP-Lmod2 (light gray bars), or GFP-Lmod2[W405*] (dark gray bars). (A) LV internal diameter in diastole. (B) Systolic performance determined by left ventricular ejection fraction (EF). (C) Western blot analysis of exogenous Lmod2 protein levels in the LV of adeno-associated virus–injected mice. Relative protein levels following normalization to total protein via Ponceau S staining are shown. Values are means ± SD; n = 6, 5, and 5 (knockout + GFP, knockout + GFP-Lmod2, and knockout + GFP-Lmod2[W405*], respectively). *P < 0.05, **P < 0.01, and ***P < 0.001, one-way ANOVA with Tukey’s post hoc test.