Alteration of performance in a mouse model of Emery–Dreifuss muscular dystrophy caused by A-type lamins gene mutation

Abstract

Autosomal Emery–Dreifuss muscular dystrophy (EDMD) is caused by mutations in the lamin A/C gene (LMNA) encoding A-type nuclear lamins, intermediate filament proteins of the nuclear envelope. Classically, the disease manifests as scapulo-humero-peroneal muscle wasting and weakness, early joint contractures and dilated cardiomyopathy with conduction blocks; however, variable skeletal muscle involvement can be present. Previously, we and other demonstrated altered activity of signaling pathways in hearts and striated muscles of Lmna^H222P/H222P mice, a model of autosomal EDMD. We showed that blocking their activation improved cardiac function. However, the evaluation of the benefit of these treatments on the whole organism is suffering from a better knowledge of the performance in mouse models. We show in the present study that Lmna^H222P/H222P mice display a significant loss of lean mass, consistent with the dystrophic process. This is associated with altered VO₂ peak and respiratory exchange ratio. These results showed for the first time that Lmna^H222P/H222P mice have decreased performance and provided a new useful means for future therapeutic interventions on this model of EDMD.

Introduction

Lamin A/C gene (LMNA) mutations cause an autosomal dominant inherited form of Emery–Dreifuss muscular dystrophy (EDMD) (1). EDMD is characterized by the clinical triad of joint contractures that begin in early childhood, slowly progressive muscle weakness and wasting initially in a humero-peroneal distribution that later extends to the scapular and pelvic girdle muscles, and cardiac involvement that manifests as poor exercise tolerance and congestive heart failure (2,3). Clinical variability ranges from early onset with severe presentation in childhood to late onset with slow progression in adulthood. Cardiac involvement usually occurs after the second decade (4). LMNA encodes the A-type nuclear lamins (5), which arise from alternative RNA splicing and are the constituents of nuclear lamina. Given the ability of mouse models to phenocopy various aspects of cardiovascular diseases, the use of mouse models has become critical to the study of cardiac biology, physiology and novel therapeutics prior to translating findings to human. We largely contributed in understanding the metabolic and physiological mechanisms regulating cardiac function and energy balance in EDMD, both in animal models and in patient’s tissues. These include abnormal regulation of the mitogen-activated protein kinase signaling cascade (6,7), altered actin dynamics (8), altered Wnt/β-catenin signaling (9), fibrosis (10), NAD signaling (11) and AKT/mammalian target of rapamyicin (mTOR) signaling (12). Despite the growing understanding of the pathogenesis of EDMD as well as the discovery of novel therapeutic for the disease (7,9–13), little is known on the overall benefit of these treatments on the performance of the animals. There is a need for more comprehensive ways of assessing disease progression and treatment efficacy. Traditional approaches to monitoring disease progression in preclinical models of muscular dystrophies are limited mostly to histological analyses and functional measurements of single fibers or muscle groups. However, none of these tests can assess systemic muscle function in a non-invasive approach consistent with clinical assessment in EDMD patients.

Figure 1

Alteration of left ventricular function in Lmna^H222P/H222P mice. (A) Graphs showing mean LVEDD, mean LVESD and FS in 3-month-old and 6-month-old male Lmna^H222P/H222P (H222P) and WT mice (n = 6). Values for each individual mouse as well SEMs of means (bars) are shown. ^**P < 0.005, ^***P < 0.0005. (B) Histological analysis of heart sections stained with hematoxylin and eosin from Lmna^H222P/H222P (H222P) and WT mice. Scale bar, 1 mm. Transthoracic M-mode echocardiographic tracings in Lmna^H222P/H222P (H222P) and WT mice. LVESD and LVEDD are indicated. (C) Sirius Red staining of cross sections of heart from 3-month-old and 6-month-old male Lmna^H222P/H222P (H222P) and WT mice. Scale bar, 25 μm.

Respiration is a product of systemic skeletal and cardiac muscle function (14). We here hypothesized that quantifying respiration during exercise would be a mean to studying systemic muscle function in a mouse model of EDMD, as it is commonly used to assess performance in humans (15). During testing, cardiac output primarily drives the associated increase in oxygen consumption (VO₂) until peak oxygen consumption (VO₂ peak) and exhaustion is achieved. Combined measurements of exercise, respiratory performance and histological marker expression therefore provide a comprehensive, reliable and powerful mean for assessing drug efficacy in a mouse model for EDMD, which in turn could help advance drug discovery.

Figure 2

Alteration of body composition in Lmna^H222P/H222P mice. (A) Graphs showing mean BW (g) in 3-month-old and 6-month-old male Lmna^H222P/H222P (H222P) (n = 16 at 3 months of age and n = 20 at 6 months of age) and WT mice. Values for each individual mouse as well SEMs of means (bars) are shown. ^*P < 0.05, ^***P < 0.0005. (B) Graphs showing mean body fat mass (g) and body fat mass relative to BW in 3-month-old and 6-month-old male Lmna^H222P/H222P (H222P) (n = 16 at 3 months of age and n = 20 at 6 months of age) and WT mice. Values for each individual mouse as well SEMs of means (bars) are shown. ^**P < 0.005, ^***P < 0.0005, ^****P < 0.00005. (C) Graphs showing mean body fat mass (g) and body fat mass relative to BW in 3-month-old and 6-month-old male Lmna^H222P/H222P (H222P) (n = 16 at 3 months of age and n = 20 at 6 months of age) and WT mice. Values for each individual mouse as well SEMs of means (bars) are shown. ^***P < 0.0005, ^****P < 0.00005.

Results

Depressed left ventricular function in Lmna^H222P/H222P mouse model

To explore the role of A-type lamins in the development of EDMD, we studied a mouse model that displays an Lmna mutation changing the histidine in position 222 into a proline (15). We used transthoracic echocardiography to determine the cardiac function of Lmna^H222P/H222P male mice. While the cardiac structure and function were not statistically different at 3 months of age, at 6 months of age, Lmna^H222P/H222P male mice have increased left ventricular diameters and decreased fractional shortening (FS) compared with wild-type (WT) animals (Fig. 1A) (8,9,16). Left ventricular dilatation in Lmna^H222P/H222P male mice at 6 months of age was demonstrated by histopathological analysis (Fig. 1B). M-mode transthoracic echocardiography showed increased left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) in Lmna^H222P/H222P mice compared with WT mice (Fig. 1B). We showed a marked increase of myocardial fibrosis in heart from Lmna^H222P/H222P male mice at 6 months of age, as observed by Sirius Red staining (Fig. 1C).

Alteration of body composition in Lmna^H222P/H222P mouse model

As the EDMD progresses further, skeletal muscle continues to be lost, which can result in weight loss. We assessed the body composition using DEXA lean/fat in Lmna^H222P/H222P mice. We observed that Lmna^H222P/H222P mice have a significant difference in body weight (BW) compared with WT mice (Fig. 2A). In 3-month-old Lmna^H222P/H222P mice, the mean of BW was 24.12 g, while from WT mice it was 26.66 g. At 6 months of age, the mean of BW was 24.94 g for Lmna^H222P/H222P mice, while it was 28.14 g for WT mice. Further complicating these issues are the changes in body composition that are linked to EDMD. We observed that Lmna^H222P/H222P mice have markedly increased body fat mass and relative body fat mass (Fig. 2B and C) and reduced body lean mass and relative body lean mass (Fig. 2B and C) compared with WT mice. The loss of lean mass was consistent with the dystrophic process (Fig. 3A and B) and the increase in adiposity could result from the decreased activity associated with the loss of activity.

Figure 3

Dystrophic skeletal muscles in Lmna^H222P/H222P mice hematoxylin-eosin staining of cross sections of diaphragm (A) and soleus (B) from 3- and 6-month-old male Lmna^H222P/H222P (H222P) and WT mice.

Alteration of physical performance in Lmna^H222P/H222P mouse model

We therefore asked whether we could assess the activity of Lmna^H222P/H222P mice. Forced exercise on a treadmill was used to measure VO₂ peak. The VO₂ peak corresponds to both the cardiovascular system’s functional limitation and the organism’s aerobic capacity. Peak oxygen consumption has been previously validated to assess prognosis in patients with heart failure (17). We showed that Lmna^H222P/H222P mice have a significant reduction in VO₂ peak, as well as relative VO₂ peak, compared with WT mice at 3 and 6 months of age (Fig. 4A). The ratio VCO₂/VO₂ is the respiratory exchange ratio (RER), which can be used to determine the proportion of carbohydrates and fatty acid utilized during an activity. Peak RER has been used as an objective parameter of effort (18). The Lmna^H222P/H222P mice have a significant decreased RER at VO₂ peak (Fig. 4B). The lower RER at VO₂ peak observed in Lmna^H222P/H222P male mice indicates a higher level of lipid metabolism when compared with WT mice. Importantly, there was a significant difference in the maximal speed and the running distance between the two genotypes (Fig. 5); thus, the decreased metabolic performance of Lmna^H222P/H222P mice resulted to a change in physical activity.

Figure 4

Alteration of maximal performance in Lmna^H222P/H222P mice. (A) Graphs showing oxygen consumption (VO2) peak (ml/h) and VO2 relative to lean mass (ml/h/g) in 3-month-old and 6-month-old male Lmna^H222P/H222P (H222P) (n = 16 at 3 months of age and n = 20 at 6 months of age) and WT mice. Values for each individual mouse as well as means (histograms) and SEMs of means (bars) are shown. ^****P < 0.00005. (B) Graphs showing RER at VO2 peak in 3-month-old and 6-month-old male Lmna^H222P/H222P (H222P) (n = 16 at 3 months of age and n = 20 at 6 months of age) and WT mice. Values for each individual mouse as well as means (histograms) and SEMs of means (bars) are shown. ^***P < 0.0005, ^****P < 0.00005.

Figure 5

Alteration of physical performance in Lmna^H222P/H222P mice. Graphs showing mean maximal speed (m/s) and distance (m) in 3-month-old and 6-month-old male Lmna^H222P/H222P (H222P) (n = 16 at 3 months of age and n = 20 at 6 months of age) and WT mice. Values for each individual mouse as well SEMs of means (bars) are shown. ^*P < 0.05, ^***P < 0.0005, ^****P < 0.00005.

We next tested whether interventions on mice could alter these parameters of effort. Given that the nicotinamide adenine dinucleotide (NAD⁺) precursor nicotinamide riboside (NR) supplemented diet was recently shown to have benefit on cardiac contractility in Lmna^H222P/H222P mice (11), we next assessed in a pilot study the NR effect on physical and metabolic performances in Lmna^H222P/H222P male mice. Since we recently reported an aberrant cardiac NAD⁺ content, we first assessed the steady-state skeletal muscle (soleus) NAD⁺ level in both male WT and Lmna^H222P/H222P mice. We showed that the NAD⁺ content was significantly lowered in hearts from Lmna^H222P/H222P mice at 6 months of age compared with WT mice, when the structure of soleus was altered (symptomatic) (Fig. 6A). We next assessed the expression level of enzymes from the salvage pathway of NAD⁺ biosynthesis by real-time polymerase chain reaction (PCR) using RNA extracted from Lmna^H222P/H222P mouse soleus at 6 months of age. There was a significant increased Nmrk2 mRNA expression in the soleus of Lmna^H222P/H222P mice compared with WT mice (Fig. 6B). We showed that feeding Lmna^H222P/H222P mice with NR, the substrate for Nmrk2, for 2 months improved significantly RER at VO₂ peak compared with chow diet-fed mice (Fig. 6C). We also reported a significant difference in the maximal speed and the distance between the NR-fed and the chow diet-fed groups (Fig. 6D). Taken together, these results showed that RER at VO₂ peak as well as physical performance are reflecting a partially restoration of the striated muscle structure and function and could therefore be used to assess future benefit of treatment in mouse model of EDMD.

Figure 6

NR supplementation leads to improvement of performance in Lmna^H222P/H222P mice. (A) Quantification of NAD⁺ content in soleus from WT (n = 6) and Lmna^H222P/H222P (n = 6) mice at 3 and 6 months of age. ^*P < 0.05. (B) Expression of Nmrk2 mRNA obtained by qPCR from WT (n = 5) and Lmna^H222P/H222P (n = 5) mice at 6 months of age. ^***P <  0.0005. (C) Graphs showing RER at VO₂ peak in 6-month-old male chow diet-fed (n = 9) or NR-fed Lmna^H222P/H222P (H222P) (n = 10). Values for each individual mouse as well as means (histograms) and SEMs of means (bars) are shown. ^**P < 0.005, ^***P < 0.0005. (D) Graphs showing mean maximal speed (m/s) and distance (m) in 6-month-old male chow diet-fed (n = 9) or NR-fed Lmna^H222P/H222P (H222P) (n = 10). Values for each individual mouse as well SEMs of means (bars) are shown. ^***P < 0.0005, ^****P < 0.00005.

Discussion

There is an important need for a more comprehensive approach to evaluate the efficacy of therapeutics for neuromuscular diseases. Although traditional measures of disease pathology are useful, they have a limited functionality given that they do not assess the whole body. Muscle disease is usually assessed by histological analysis and circulating markers of myofiber injury or functional assessments of isolated muscles. Our findings strongly suggest that non-invasive protocols for assessing exercise performance in Lmna^H222P/H222P mice quantifying several parameters of respiration and exercise are useful to assess the dystrophic pattern in EDMD. Exercise performance (e.g. 6 min walk test) was recently used as the primary end point to assess the benefit of p38 inhibitor ARRY-797 (7) in patients with cardiomyopathy caused by LMNA mutation (ClinicalTrials.gov Identifier: NCT02057341). This clinical trial showed that patients receiving ARRY-797 performed better, with a mean change from baseline, of 69 meters.

Measures of respiratory performance during exercise have been established for other animal model of muscular dystrophy (19), but are lacking for mouse models of EDMD mice. We showed that respiratory performance was impaired in Lmna^H222P/H222P mice. Our findings illustrate the usefulness of respiratory performance in quantifying the dystrophic phenotype in EDMD, as differences in several exercise parameters (e.g. VO₂ peak, RER) were detected even before detection of dystrophic pattern by histological analysis. Thus, quantifying respiratory performance during exercise is in some way a more sensitive means for assessing muscular dystrophy in a mouse model for EDMD. The unique patterns of VO₂ peak and RER in exercising Lmna^H222P/H222P mice identified novel and very useful markers for assessing exercise performance. Our findings would benefit the investigational drugs that could correct loss of skeletal muscle and could ameliorate performance in EDMD. Along these lines, we showed that interventional treatment using NR significantly improved RER at VO₂ peak in Lmna^H222P/H222P mice.

The RER is defined as the CO₂ produced/O₂ consumed. This ratio increases with the exercise intensity and is commonly used to indirectly determine the relative contribution of carbohydrate and lipids to overall energy expenditure. In Lmna^H222P/H222P mice, we showed a decreased RER compared with WT mice, indicating greater reliance on fatty acid oxidation. It has long been recognized that substrate metabolism is altered in the heart that undergoes pathological conditions (20). We recently reported a marked decrease of pyruvate to lactate exchange kinetics in the heart muscle from Lmna^H222P/H222P mice (21). Moreover, we recently reported a cardiac muscle NAD⁺ depletion in Lmna^H222P/H222P mice (9), suggesting a decreased activity of the pyruvate dehydrogenase complex and a reduced production of acetyl coenzyme A from carbohydrates. It would be interesting to extensively perform metabolomics studies in these mice to address the primary metabolic defect, which lead to decline in the function.

Assessing respiration (VO₂ and VCO₂) during exercise greatly increases the overall understanding of disease pathology, as it examines the combination of cardiac and skeletal muscle functions. Taken together, our study suggests that the use of respiratory performance during exercise to quantify changes in the dystrophic phenotype is a good complement to histological markers. Indeed, it can provide non-invasive markers for assessing EDMD disease progression, which is not always reflected histologically. The phenotypic changes observed in Lmna^H222P/H222P mice can now be used to guide future interventional therapies to assess their beneficial effect.

Materials and Methods

Mice

Lmna^H222P/H222P mice (16) were fed chow and housed in a disease-free barrier facility at 12 h/12 h light–dark cycles. All animal experiments were approved by the French Ministry of Health at the Center for Research in Myology for the Care and Use of Experimental Animals. The animal experiments were performed according to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Chow supplemented with 0.25% NR (400 mg/kg) (Chromadex), as previously reported (11), was formulated by Scientific Animal Food and Engineering.

Echocardiography

Mice were anesthetized with 0.75% isoflurane in O₂ and placed in a heating pad. Transthoracic echocardiography was performed using an ACUSON 128XP/10 ultrasound with an 11 MHz transducer applied to the chest wall. All parameters were measured by a ‘blinded echocardiographer’ unaware of the genotype or treatment of the mice, performed in 2D mode and M-mode in triplicate.

Histology

Frozen pieces of soleus and diaphragm were mounted in Tissue-Tek (Fisher Scientific, Hampton, New Hampshire, USA) and 10 μm sections cut on a cryostat. Sections were stained with hematoxylin and eosin for histological analysis. Representative sections were photographed using a Microphot SA (Nikon, Minato, Tokyo, Japan) light microscope attached to a Spot RT Slide camera (Diagnostic Instruments, Sterling Heights, Michigan, USA). Images were processed using Adobe Photoshop CS (Adobe Systems, San Jose, California, USA).

Quantitative reverse transcription PCR (qPCR) analysis

Total RNA was extracted using the RNeasy Mini kit (Qiagen, Hilden, Germany). cDNA was synthesized using the SuperScript III synthesis system according to the manufacturer’s instructions (Invitrogen, Carlsbad, California, USA). Real-time qPCR reactions were performed with SYBR Green I Master mix (Roche) using with the LightCycler 480 (Roche, Basel, Switzerland). Relative levels of mRNA expression calculated using the ΔΔC_T method were normalized by comparison to housekeeping mRNA.

NAD⁺ concentration quantification

Fresh mouse tissues were homogenized and processed according to previous report (11). The concentration of soleus NAD⁺ was determined using a spectrophotometric assay as described previously (11). Absorption measurements were carried out in 96-well plates by spectrophotometry (Tecan).

Weight and body composition

The weight of the mice and their body composition (lean mass and fat mass) were measured throughout the protocol period. For body composition we used a nuclear magnetic resonance system (LF90II, Bruker, Germany).

Evaluation of the physical performance

Each mouse was evaluated on a one-way treadmill equipped with an indirect calorimetry system (Phenomaster, TSE, Germany). The performance evaluation protocol consisted of increasing the speed by 0.01 m.s⁻¹ every 15 s. Oxygen consumption (VO₂), carbon dioxide production (VCO₂) and RER (RER = VCO₂/VO₂) were recorded every second. Oxygen consumption peak (VO_{2 peak} in ml.h⁻¹) was set as the highest VO₂ value achieved during the test. We developed an algorithm based on the Savitzky–Golay principle (22) to automate the analysis of calorimetric data by smoothing the noisy data. The maximum speed reached by each mouse was recorded. The maximum speed corresponds to the speed at which the mouse can no longer run on the treadmill. All protocols were conducted for each mouse until exhaustion. Exhaustion was defined as the time when the animal ‘preferred’ to receive electric stimulation through the grid installed at the beginning of the treadmill rather than continue the race. The intensity of the electric stimulation was 1.2 mA during 1 s, with 1 s latency. Before each protocol, there was a 3 min acclimatization on the treadmill at low speeds from 0.01 to 0.05 m.s⁻¹. All these performance measurements were realized in the ‘Performance & Metabolism in Mice’ facility (PMM, EA7329, Paris Descartes University, Paris, France).

Statistics

Statistical analyses were performed using GraphPad Prism software. Statistical significance between groups of mice was analyzed with a corrected parametric test (Welch’s t-test), with a value of P < 0.05 being considered significant. To validate results, we performed a non-parametric test (Wilcoxon–Mann–Whitney test). Values are represented as means ± standard errors of mean (SEM).

Acknowledgements

The authors gratefully acknowledge ChromaDex for providing NR.

Conflict of Interest statement. None declared.

Funding

Association Française contre les Myopathies; Institut National de la Santé et de la Recherche Médicale; Agence Nationale de la Recherche (ANR-17-CE17-0015-03); Région Ile de France (to P.N.); labex GR-Ex (to P.N.). The labex GR-Ex, reference ANR-11-LABX-0051, is funded by the program ‘Investissements d’avenir’ of the French National Research Agency (ANR-11-IDEX-0005-02).