In vivo elongation of thin filaments results in heart failure

Abstract

A novel cardiac-specific transgenic mouse model was generated to identify the physiological consequences of elongated thin filaments during post-natal development in the heart. Remarkably, increasing the expression levels in vivo of just one sarcomeric protein, Lmod2, results in ~10% longer thin filaments (up to 26% longer in some individual sarcomeres) that produce up to 50% less contractile force. Increasing the levels of Lmod2 in vivo (Lmod2-TG) also allows us to probe the contribution of Lmod2 in the progression of cardiac myopathy because Lmod2-TG mice present with a unique cardiomyopathy involving enlarged atrial and ventricular lumens, increased heart mass, disorganized myofibrils and eventually, heart failure. Turning off of Lmod2 transgene expression at postnatal day 3 successfully prevents thin filament elongation, as well as gross morphological and functional disease progression. We show here that Lmod2 has an essential role in regulating cardiac contractile force and function.

Fig 1. High levels of Lmod2 protein expression in Lmod2-TG hearts correspond with longer thin filaments.

(A) Analysis of immunoblots of left ventricular (LV) tissue extracts of P1, P7, P30 and P60 mice probed with anti-Lmod2 antibodies. Lmod2 transgene expression is highly elevated in Lmod2-TG (black) when compared with endogenous Lmod2 levels in NTG (white) at all time points. Data are presented as the relative expression level of Lmod2 in Lmod2-TG animals when compared with NTG for each time point. (n = 4–8; Error bar = SEM; two-tailed unpaired t-test). (B) Representative immunoblot (Lmod2 and c-Myc at ~80.kDa) and Ponceau S-stained membrane (PonS) of P7 LV extracts. (C) Quantitative measurements of cardiac thin filament length (TFL) in P1, P7, P30 and P60 mice. TFL of Lmod2-TG hearts (black) are 3–10% longer when compared to those of NTG (white). Gray bars represent comparison of NTG TFL between each time points, and black bars represent those of Lmod2-TG (n = 4–8; Error bar = SEM; two-way ANOVA with Tukey test). (D) Representative fluorescently-conjugated phalloidin staining of LV tissue from P7 NTG and two Lmod2-TG (TG #1, #2) mice (arrows show thin filament pointed ends; Z shows Z-disc/thin filament barbed ends). Scale bar = 1 μm.

Fig 2. All chambers of Lmod2-TG hearts are enlarged with extensive interstitial fibrosis by post-natal day 30 (P30).

(A) Atrial and ventricular chambers in Lmod2-TG hearts are larger than those in Lmod2 transgene-null (NTG) hearts. Scale bar = 1 mm. (B) Trichrome histological stain indicates the existence of fibrosis within the ventricular myocardium of Lmod2-TG hearts. (C) Quantification of Picrosirius Red-stained ventricular tissue reveals a significantly higher level of interstitial fibrosis in Lmod2-TG (black), when compared with NTG (white) (n = 6–8; Error bar = SEM; two-tailed unpaired Welch’s t-test).

Fig 3. Real-time quantitative PCR (qPCR) of fetal genes in NTG and Lmod2-TG shows significant disease progression at P30.

Re-activation of the fetal gene program is indicative of a diseased state of the heart; hence, qPCR of Lmod2-TG mice at P7 (top panels) and P30 (bottom panels) were examined using mRNA extracted from LV tissue. Though not statistically significant, relative expression levels of 3 out of 5 molecular heart disease markers (Acta1, Nppa and Nppb) are trending higher in Lmod2-TG (black) when compared to NTG (white) at P7. At P30, relative expression levels of all disease markers examined are significantly altered (n = 4–6; Error bar = SEM; two-tailed unpaired Welch’s t-test).

Fig 4. Ultrastructural analysis of Lmod2-TG heart indicates extensively disrupted myofibrillar organization.

Transmission electron micrograph of P30 LV tissue reveals disorganized myofibrillar cytoarchitecture, with out-of-register sarcomeres in Lmod2-TG hearts (representative images from n = 3 per genotype; scale bar = 2 μm).

Fig 5. Overexpression of Lmod2 negatively affects contractile force at the single myofiber level.

At P15, Lmod2-TG trabeculae fibers (black) show a significant drop in raw force measured at sarcomere lengths of 1.95 μm (A) and 2.10 μm (B) when compared with NTG fibers (gray), indicative of defective contractile force generation. Passive tension average (C), as well as the calcium sensitivity (EC₅₀) and myofilament cooperativity (Hill coefficient) (S4 Table) of P15 Lmod2-TG and NTG littermates are not significantly different from each other. By P60, however, passive tension average (C) in Lmod2-TG (red) is drastically higher than those of NTG (blue), most likely due to extensive interstitial fibrosis. N = 1 fiber per animal from 7–9 animals. Error Bar = SEM.

Fig 6. Lmod2 transgene expression off: Lmod2 contributes to in vivo cardiac function.

(A) Analysis of immunoblots of P60 LV tissue extracts probed with anti-Lmod2 antibodies. Lmod2-cre mice, Lmod2-TG with Lmod2 transgene expression turned off, have Lmod2 protein levels similar to NTG. (B) Lmod2-cre mice have thin filament lengths similar to those of NTG. (C) Pressure-Volume Loops analysis reveals that the systolic (ESPVR, end-systolic pressure-volume relationship) and diastolic (EDPVR, end-diastolic pressure-volume relationship) dysfunctions observed in Lmod2-TG mice can be prevented by turning Lmod2 transgene expression off. N = 4–6; Error bar = SEM; non-parametric one-way ANOVA with Tukey test.