Lmod2 piggyBac mutant mice exhibit dilated cardiomyopathy

Abstract

Background: Leiomodin proteins, Lmod1, Lmod2 and Lmod3, are key regulators of the thin filament length in muscles. While Lmod1 is specifically expressed in smooth muscles, both Lmod2 and Lmod3 are expressed in striated muscles including both cardiac and skeletal muscles. We and others have previously shown that Lmod3 mainly function in skeletal muscles and the mutant mice display disorganized sarcomere. Lmod2 protein has been found to act as an actin filament nucleator in both cell-free assays and in cultured rat and chicken cardiomyocytes.

Results: To better understand the function of Lmod2 in vivo, we have identified and characterized a piggyBac (PB) insertional mouse mutant. Our analysis revealed that the PB transposon inserts in the first exon of the Lmod2 gene and severely disrupts its expression. We found that Lmod2 (PB/PB) mice exhibit typical dilated cardiomyopathy (DCM) with ventricular arrhythmias and postnatal lethality. Electron microscope reveals that the Lmod2 (PB/PB) hearts carry disordered sarcomere, disarrayed thin filaments, and distorted intercalated discs (ICDs). Those ICDs display not only decreased convolutions, but also reduced electron-dense staining, indicating less ICDs component proteins in Lmod2 (PB/PB) hearts. Consistent with the phenotype, the expression of the ICD component genes, β-catenin and Connexin43, are down-regulated.

Conclusions: Taken together, our data reveal that Lmod2 is required in heart thin filaments for integrity of sarcomere and ICD and deficient mice exhibit DCM with ventricular arrhythmias and postnatal lethality. The Lmod2 (PB/PB) mutant offers a valuable resource for interrogation of pathogenesis and development of therapeutics for DCM.

Keywords: Dilated cardiomyopathy; Intercalated discs; Leiomodin2 mutant; Sarcomere.

Fig. 1

PB transposon disrupts Lmod2 expression. a Schematic representation of the Lmod2 transcription unit and the position of PB insertion. Coding and untranslated region are depicted as black and white boxes, respectively. b Quantitative RT-PCR analysis of Lmod2 mRNA from heart of 3-week-old male and female mice with indicated genotypes, n = 3. Arrows PCR primers

Fig. 2

Tmod1 and Lmod3 Expression in Lmod2 mutant mice. a mRNA level of Tmod1 is not changed in Lmod2^PB/PB hearts compared with wild-type control. b Lmod3 protein level is also not changed in Lmod2^PB/PB mice as shown here in western blotting and statistics

Fig. 3

Lmod2^PB/PB mice exhibit postnatal lethality and underweight in surviving males. Survival a and growth b curves of male and female Lmod2^PB/PB mice compared with Lmod2^PB/+ and Lmod2^+/+ mice

Fig. 4

Lmod2^PB/PB hearts display dilated cardiomyopathy defects. 25-day old Lmod2^PB/PB mice were examined for cardiac morphology and functions. a Longitudinal (upper) and transverse (lower) H&E stain sections of paraffin-embedded hearts (Scale bar 1 mm). b, c, e Echocardiography analysis of Lmod2^PB/PB hearts in comparison to wild-type control n = 5. b Higher LV end diastolic and systolic diameter. LVID left ventricular diameter. c Thinner LV anterior and posterior wall. LVPW left ventricular posterior wall; LVAW left ventricular anterior wall. d Morphometric ratios (heart/body weight) of both male and female Lmod2^PB/PB mice are significantly increased. e Reduced ejection fraction and fractional shortening values. f Quantitative RT-PCR analysis of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) revealed increased expression of the heart hypertrophy and heart failure biomarkers n = 3. LV left ventricle; d diastolic; s: systolic. g, h Electronic Cardiogram revealed lengthening QTc value of Lmod2^PB/PB mice in comparison to controls

Fig. 5

ICD and sarcomere defects of Lmod2^PB/PB heart fibers and reduced expression of ICDs proteins. a, b Longitudinal sections of 25d paraffin-embedded hearts stained with H&E. c–h Representative electron micrographs of cardiac LV tissue from 25 days old Lmod2^+/+ (c, e, g) and Lmod2^PB/PB (e, f, h) mice. Sarcomeres (S) are shortened and disorganized in Lmod2^PB/PB myocardium (c, d). Z-discs (Z) and M-lines (M) are disorganized and unrecognizable, respectively, in Lmod2^PB/PB myocardium (e, f). Reduced convolution and electron dense of ICD (arrow) in Lmod2^PB/PB myocardium (g, h). i Area and perimeter of the area between the muscle fibers are increased in Lmod2 ^PB/PB hearts compared with wild-type control n = 8. j Statistics of sarcomere length in Lmod2^PB/PB myocardium n = 20. k Quantitative RT-PCR of major intercalated discs (ICD) genes n = 3. Scale bars 200 μm in b, 1 μm in d, 500 μm in f, h