Heterozygous Mylk3 Knockout Mice Partially Recapitulate Human DCM With Heterozygous MYLK3 Mutations

Abstract

Backgrounds: Recent studies identified heterozygous variants in MYLK3 gene that encodes cardiac myosin light chain kinase (cMLCK) are related to familial dilated cardiomyopathy (DCM) for the first time. Autosomal dominant traits suggest that pathogenesis of DCM could be related to heterozygous MYLK3 loss-of-function variants (haploinsufficiency). We previously generated and examined homozygous Mylk3 knockout mice that lead to heart failure. It had yet to be examined whether heterozygous Mylk3 knockout mice represent a DCM-like phenotype. Methods and Results: Heterozygous knockout (Mylk3 ^wild/-) mice were examined regarding cardiac function, heart histology and expression of cMLCK protein and mRNA relative to age-matched wild-type controls (Mylk3 ^wild/wild). At 4 months of age, cardiac contractility in heterozygous knockout mice was reduced with percent fractional shortening of 23.3 ± 1.2% compared to 30.1 ± 1.8% in control (Mylk3 ^wild/- vs. Mylk3 ^wild/wild, n = 9 each). In 4-month-old heterozygous knockout hearts, expression of cMLCK mRNA was expectedly reduced by almost half, however, protein expression was reduced by approximately 75% relative to the control wild-type (Mylk3 ^wild/- vs. Mylk3 ^wild/wild, n = 9 each). Isolated ventricular cardiomyocytes from heterozygous knockout mice were larger with increase of short-axis length relative to the cardiomyocytes from control mice. However, increase of heart failure markers as well as interstitial fibrosis were not evident in heterozygous knockout mice compared to controls. Conclusion: Heterozygous Mylk3 knockout mice show mild reduction of cardiac contractility by 4 months of age, and proteins reduced by approximately 75% relative to the control wild-type mice. These mice partly resemble human with the heterozygous MYLK3 mutation, but the reduction in cardiac contractility was milder.

Keywords: animal model of human disease; genetic dilated cardiomyopathy; heart; heterozygous knockout; kinase.

FIGURE 1

Mild heart enlargement and reduced contractile function in heterozygous Mylk3 knockout mice. (A) By mating Mylk3^wild/- mice, Mylk3^wild/wild, Mylk3^wild/-, and Mylk3^-/- were generated. Representative hearts dissected from three groups of mice at 4 months of age. Bar = 2 mm. (B) HW/BW (mg/g) at 3 weeks and 4 months of age. (C,D) Representative images of M-mode ultrasound and echocardiographic indices of Mylk3^wild/wild and Mylk3^wild/- mice at 4 months of age. Representative tracing of LVPV measurements (E) and summarized data (F) from Mylk3^wild/wild and Mylk3^wild/- mice at 4 months of age. Data are expressed as mean ± SEM, with or without individual data that are presented by black circles. P values are indicated in panel B, or ^∗ indicates P < 0.05. HW/BW, heart weight/body weight ratio; ED, end-diastolic; ES, end-systolic; %FS, % left ventricular fractional shortening; PW, posterior wall; ESV, end-systolic volume; EDV, end-diastolic volume; ESP, end-systolic pressure; EDP, end-diastolic pressure; dP/dt max, peak rate of pressure rise; dP/dt min, peak rate of pressure decline; PRSW, preload recruitable stroke work; Ees, end-systolic elastance; EDPVR slope, end-diastolic PV relation slope; Tau, relaxation time constant.

FIGURE 2

Enlarged cardiomyocytes with reduced contractility and impaired Ca²⁺-handling in heterozygous Mylk3 knockout mice. (A) Representative images of Picro Sirius Red-stained transverse sections of the hearts. (B) Representative images of cardiomyocytes, short axis (μm), long axis (μm), cell area (μm²) and ratio of short vs. long axis of cardiomyocytes isolated at 4 months of age (Mylk3^wild/wild, n = 371; Mylk3^wild/-, n = 351 from n = 3 mice each). Bars = 100 μm. (C) Measurements of cardiac contraction and simultaneous Ca²⁺ transients in isolated cardiomyocytes (Mylk3^wild/wild, n = 171; Mylk3^wild/-, n = 158 from n = 3 mice each). Data are expressed as mean ± SEM. ^∗P < 0.05. +dL/dt (speed of contraction); –dL/dt (speed of relaxation).

FIGURE 3

Reduced cMLCK proteins and MLC2 phosphorylation in heterozygous Mylk3 knockout hearts. (A) Representative Western blotting of cMLCK, pMLC2v, total MLC2v and GAPDH using isolated hearts from Mylk3^wild/wild and Mylk3^wild/- mice at 4 months of age. The cropped blots are used in the figure. (B) Fold difference of cMLCK, pMLC2v and total MLC2v normalized to GAPDH with the value of Mylk3^wild/wild defined as 1 (n = 9 each). (C) Real-time RT-PCR demonstrates relative expression of cMLCK, atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), skeletal MLCK, smooth muscle MLCK, and Dapk3 mRNA normalized to β-actin with the value in Mylk3^wild/wild defined as 1. Data are expressed as mean ± SEM. ^∗P < 0.05. ANF, atrial natriuretic factor; BNP, brain natriuretic peptide; Sm, smooth muscle.

FIGURE 4

Altered responses to UPS inhibitors and cycloheximide between heterozygous knockout and wild-type adult cardiomyocytes. (A) Representative Western blotting of cMLCK and total MLC2v using adult cardiomyocytes isolated from Mylk3^wild/wild and Mylk3^wild/- mice at 4 months of age at time 0 and 48 h of incubation with or without UPS inhibitors. The cropped blots are used in the figure. (B) Fold difference of cMLCK relative to total MLC2v with the value of Mylk3^wild/wild at time 0 defined as 1 (n = 4 each). (C) Representative immunostaining of cMLCK and MLC2v in the heart sections (n = 3 each). (D) Representative immunostaining of cMLCK and Z-disk protein α-actinin2, and cMLCK and M-line protein myomesin in the heart sections. Data are expressed as mean ± SEM. ^∗P < 0.05. Bars = 20 μm.