Cardiac-specific knockout of Lmod2 results in a severe reduction in myofilament force production and rapid cardiac failure

Abstract

Leiomodin-2 (Lmod2) is a striated muscle-specific actin binding protein that is implicated in assembly of thin filaments. The necessity of Lmod2 in the adult mouse and role it plays in the mechanics of contraction are unknown. To answer these questions, we generated cardiac-specific conditional Lmod2 knockout mice (cKO). These mice die within a week of induction of the knockout with severe left ventricular systolic dysfunction and little change in cardiac morphology. Cardiac trabeculae isolated from cKO mice have a significant decrease in maximum force production and a blunting of myofilament length-dependent activation. Thin filaments are non-uniform and substantially reduced in length in cKO hearts, affecting the functional overlap of the thick and thin filaments. Remarkably, we also found that Lmod2 levels are directly linked to thin filament length and cardiac function in vivo, with a low amount (<20%) of Lmod2 necessary to maintain cardiac function. Thus, Lmod2 plays an essential role in maintaining proper cardiac thin filament length in adult mice, which in turn is necessary for proper generation of contractile force. Dysregulation of thin filament length in the absence of Lmod2 contributes to heart failure.

Keywords: Actin-thin filaments; Cardiomyopathy; Sarcomere.

Figure 1.. Knockout of Lmod2 results in impaired left ventricle systolic performance.

(A-E) Echocardiography analysis of vehicle (black bars) or tamoxifen (white bars) treated mice. (A) Ejection fraction; (B) Left ventricle internal dimension in diastole; (C) Left ventricle anterior wall thickness in diastole; (D) Left ventricle posterior wall thickness in diastole; and (E) Eccentricity index, which equals the left ventricle internal dimension in diastole divided by the sum of the anterior and posterior wall thicknesses. All values represent means ± SD; n = 8–12. (F) Immunoblot of LV lysate from mice 7 days after the first injection of vehicle (−) or tamoxifen (+). Lysate was probed with antibodies to Lmod2 and β-tubulin.

Figure 2.. Cardiac thin filament length is reduced in the Lmod2 cKO mice.

(A) Representative images of immunofluorescence staining of LV free wall in vehicle and tamoxifen treated Lmod2^fl/fl;MCM^+/− mice. F-actin is in green and α-actinin, which marks the Z-disc, is in red in the merged images. The locations of thin filament barbed (arrowheads) and pointed (arrows) ends are indicated. Scale bar = 2 μm. Examples of uniform (1) and non-uniform (2) thin filaments upon tamoxifen treatment are shown. (B) Mean focus values, which represents the uniformity of thin filament lengths within each sarcomere (the smaller the number the more uniform the filament lengths). (C) Thin filament length in LV free wall of vehicle (black bars) and tamoxifen (white bars) treated mice. All values are means ± SD; n = 8–12. (D) Thin filament length plotted against sarcomere length for Lmod2^fl/fl;MCM^+/− mice treated with vehicle (black circles) or tamoxifen (grey circles). The slopes of the two linear regression lines are significantly non-zero (p < 0.0001) and not significantly different from each other. The y-intercept of the TAM-treated linear regression line is significantly lower (p < 0.0001) than the control, indicating thin filaments are shorter when sarcomere length is taken into account.

Figure 3.. The levels of thin filament components are significantly reduced in the LV of Lmod2 cKO mice.

Immunoblot analysis of LV proteins in whole cell lysate (top panel) or insoluble fraction (bottom panel) of Lmod2^fl/fl;MCM^+/− mice treated with vehicle (black bars) or tamoxifen (white bars). (Left) Single representative blots; (Right) Mean relative protein levels following normalization to total protein via Ponceau S staining. Error bars indicate the SD, n = 5–8.

Figure 4.. Loss of Lmod2 reduces calcium-activated force and alters calcium sensitivity in cardiac trabeculae.

Force-Ca²⁺ relationship of cardiac trabeculae at short (1.95 μm-A) and long (2.25 μm-B) sarcomere lengths. The insets are graphs of the mean EC₅₀ of calcium activation. Maximum calcium-activated force at sarcomere lengths 1.95 μm (C) and 2.25 μm (D). (E) Change in maximum force as sarcomere length increases. All values are means ± SEM; n = 5–6.

Figure 5.. Total phosphorylation of thin filament regulatory proteins is not significantly changed.

(A) Representative gel of LV lysate stained for total phosphoprotein content. (B) Corresponding Coomassie blue stain for total protein. (C) Mean phosphoprotein levels normalized to total protein for mice treated with vehicle (black bars) or tamoxifen (white bars). Error bars indicate the SD, n = 5–6.

Figure 6.. Calcium activated contractile force is reduced in cardiomyocytes isolated from Lmod2 cKO mice.

(A) Force-Ca²⁺ relationship of cardiac cells at a sarcomere length of 2.25 μm. The inset is a graph of mean EC₅₀ of calcium activation. (B) Mean force at maximum calcium activation at 2.25 μm. Error bars indicate ± SEM; n = 27–35 cells from 5–6 animals. (C) Maximum calcium-activated force at various sarcomere lengths. n = 15 cells from 5 WT animals and 27 cells from 6 cKO animals.

Figure 7.. Thin filament length and cardiac function negatively correlate with tamoxifen dose; cardiac function positively correlates with thin filament length.

(A) Relative Lmod2 levels, thin filament (TF) length, ejection fraction (EF) and maximal activated force (FMax) plotted against the dose of tamoxifen (TAM) injected. 0 mg/kg are mice injected with vehicle alone. Data were fit using linear regression. All values are means ± SD; n = 3–13 animals. (B) Ejection fraction (EF) and maximal calcium activated force (FMax) plotted against thin filament length. n = 3–13 animals.