Effect of genetic background on the cardiac phenotype in a mouse model of Emery-Dreifuss muscular dystrophy

A-type lamins gene (LMNA) mutations cause an autosomal dominant inherited form of Emery-Dreifuss muscular dystrophy (EDMD). EDMD is characterized by slowly progressive muscle weakness and wasting and dilated cardiomyopathy, often leading to heart failure-related disability. EDMD is highly penetrant with poor prognosis and there is currently no specific therapy available. Clinical variability ranges from early onset with severe presentation in childhood to late onset with slow progression in adulthood. Genetic background is a well-known factor that significantly affects phenotype in several mouse models of human diseases. This phenotypic variability is attributed, at least in part, to genetic modifiers that regulate the disease process. To characterize the phenotype of A-type lamins mutation on different genetic background, we created and phenotyped C57BL/6JRj-LmnaH222P/H222P mice (C57Lmnap.H222P) and compared them with the 129S2/SvPasCrl-LmnaH222P/H222P mice (129Lmnap.H222P). These mouse strains were compared with their respective control strains at multiple time points between 3 and 10 months of age. Both contractile and electrical cardiac muscle functions, as well as survival were characterized. We found that 129Lmnap.H222P mice showed significantly reduced body weight and reduced cardiac function earlier than in the C57Lmnap.H222P mice. We also revealed that only 129Lmnap.H222P mice developed heart arrhythmias. The 129Lmnap.H222P model with an earlier onset and more pronounced cardiac phenotype may be more useful for evaluating therapies that target cardiac muscle function, and heart arrhythmias.


Introduction
Emery-Dreifuss muscular dystrophy (EDMD) is characterized by the clinical triad of i/slowly progressive muscle weakness and wasting in a scapulo-humeroperoneal distribution; ii/early contractures of the elbows, ankles, and posterior neck; and iii/cardiac conduction defects associated with dilated cardiomyopathy [1]. LMNA mutations encoding nuclear A-type lamins are responsible for the autosomal forms of EDMD [2]. Genetically engineered mouse models of EDMD have brought valuable insights in our understanding of the molecular mechanisms of the disease [3]. They have been instrumental in the identification of signaling pathways responsible for the cardiac dysfunction and have provided invaluable tools for proposing novel treatment for this disease [4][5][6][7][8][9][10]. The knock-in Lmna p.H222P/H222P mice mouse model carrying the LMNA p.H222P mutation previously described in patient with classical form of EDMD [11], recapitulates all the features of EDMD [12]. In particular, Lmna H222P/H222P mice developed a dilated cardiomyopathy associated with cardiac conduction defects [11,13].
Lmna H222P/H222P mice were generated using a C57BL/6 background, an inbred strain and useful reductionist tool to study effects of single gene mutations, However in the clinic, LMNA mutations show a strong degree of pleiotropy and therefore understanding how genetic background influences this is likely to prove important. It is known that phenotypic variation that arises due to the influence of genetic background can be of importance to genetically engineered mouse models [14,15]. More recently, it has been showed that the genetic background was a determining factor for the severity of muscular dystrophy in a mouse model of Duchenne muscular dystrophy [16,17]. To investigate the effect of genetic background on the cardiac involvement of EDMD, we have backcrossed Lmna p.H222P/H222P mice to different pure genetic background, C57BL/6JRj (C57 Lmna p.H222P ) and 129S2/SvPasCrl (129 Lmna p.H222P ). We found that cardiac involvement was more pronounced in the 129 Lmna p.H222P mouse strain compared with the C57 Lmna p.H222P mouse strain, suggesting that genetic background significantly modifies the severity of dilated cardiomyopathy linked to LMNA mutations.

Genetic characterization of mouse strains
To assess the genetic background of the C57 Lmna p.H222P and 129 Lmna p.H222P mouse strains, we used a marker-assisted selection protocol (MASP) based on genome-wide analysis of genetic polymorphisms, which allows the discrimination between different strains of mice [18]. We used simple sequence length polymorphism (SSLP) that correspond to short cytosine, adenine nucleotide tandem repeats (CA)n. The number n of (CA) repeats is specific for C57BL/6JRj or 129S2/SvPasCrl strains [19]. We screened for 50 SSLPs, covering the entire mouse genome, on all four mouse strains (Table S1). We observed that all the 50 SSLPs were homozygous and matched in length for both C57BL/6JRj and C57 Lmna p.H222P mice (Fig. 1A). Besides, we found that 48 of the 50 SSLPs were homozygous and matched in size in 129 Lmna p.H222P mice compared with 129S2/svPasCrl mice (Fig. 1A). Furthermore, one SSLP (D16-Mit-57a), located on chromosome 16, was heterozygous with one allele from C57BL/6JRj strain and the second from 129S2/SvPasCrl strain (Fig. 1B). Moreover, one SSLP (D10-Mit-11) in 129 Lmna p.H222P mice, located on chromosome 10, was homozygous for C57BL/6JRj strain. Hence, the C57 Lmna p.H222P mouse strain was entirely derived on C57BL/6JRj genetic background (Fig. 1C). Moreover, 97% of the 129 Lmna p.H222P strain was similar to 129S2/svPasCrl strain and the other 3% was similar to C57BL/6JRj strain (Fig. 1C).

Body weight
The loss of body weight characterizes the progression of the disease in Lmna H222P/H222P mice [12]. To follow the time course of the disease of the 129 Lmna p.H222P and C57 Lmna p.H222P mice, we weighed mice every month from 3 to 10 months of age. The 129 Lmna p.H222P mice started to lose weight from 6 months of age compared with 129S2/svPasCrl mice ( Fig. 2A). The C57 Lmna p.H222P mice started to lose weight at 7 months of age compared with C57BL/6JRj mice ( Fig. 2A). The median survival of the 129 Lmna p.H222P mice was significantly shorter compared to the C57 Lmna p.H222P mice, p < 0.001 (Fig. 2B). The median survival of the 129 Lmna p.H222P mice was 7 months of age, while the median survival of C57 Lmna p.H222P mice was 10 months. (Fig. 2B). The weight loss was correlated with decreased lifespan for both 129 Lmna p.H222P and C57 Lmna p.H222P mice. The coefficient determination R 2 between weight and percentage of survival was 0.89 for 129 Lmna p.H222P mice and 0.98 for C57 Lmna p.H222P mice (Fig. 2C). These results suggested that the phenotype of 129 Lmna p.H222P mice was more severe than for the C57 Lmna p.H222P mice.

Cardiac structure and function
Cardiac structure and function were determined by echocardiography in the four different groups of mice. Compared with 129S2/ svPasCrl mice, 129 Lmna p.H222P mice had significantly increased leftventricular end systolic diameter, starting at 4 months of age ( Fig. 3A and C, Table S2). The left-ventricular end diastolic diameters started to be significantly increased in 129 Lmna p.H222P mice at 8 months of age ( Fig. 3A and B, Table S2). Compared with C57BL/6JRj mice, C57 Lmna p.H222P mice had significantly increased left-ventricular end systolic and left-ventricular end diastolic diameters at 6 months of age ( Fig. 3A and C, Table S2). We observed a statistically significant decreased fractional shortening (FS) for 129 Lmna p.H222P mice compared with wild type 129S2/svPasCrl animals at 4 months of age (Fig. 3D, Table S2), while it was noticeable at 5 months of age for C57 Lmna p.H222P mice. Taken together, these results showed a more severe cardiac phenotype in the 129 Lmna p.H222P mice compared with the C57 Lmna p.H222P animals.

Conduction defects and arrhythmias
Given that conduction defects and arrhythmias have been previously described in Lmna H222P/H222P mice [12,13], we set out to assess of the cardiac electrical conduction disturbances (Fig. 4A) in the four different groups of mice. The PR interval and the QRS complex duration were extended, without reaching significance (Table S4), in 129 Lmna p.H222P mice and C57 Lmna p.H222P mice compared with 129S2/svPasCrl mice and C57BL/6JRj mice, respectively ( Fig. 4B and C). Both PR interval and QRS complex increases were more pronounced in the 129 Lmna p.H222P mice than in the C57 Lmna p.H222P mice, with an earlier onset in 129 Lmna p.H222P mice (Table S3). The RR interval was  significantly prolonged in the 129 Lmna p.H222P mice at 3, 5 and 6 months of age compared with 129S2/svPasCrl mice (Fig. 4D, Table S3). Similarly, RR interval was also lengthened in the C57 Lmna p.H222P mice at 6 months compared with C57BL/6JRj mice (Fig. 4D, Table S3). Furthermore, ECG analyses revealed that neither C57BL/6JRj mice nor C57 Lmna p.H222P mice develop heart arrhythmias at 3 or 5 months of age (Fig. 4E,   F, 4G, 4H). However, 129 Lmna p.H222P mice developed heart arrhythmia (supraventricular premature contractions and sino ventricular blocks) at 3 ( Figure 4I) and 5 months of age (Fig. 4J). We noted that 129S2/ svPasCrl mice did not have heart arrhythmia at 3 months of age ( Fig. 4K) but developed some by 5 months of age ( Figure 4L). Taken together these results showed a higher susceptibility to conduction defects in 129 Lmna p.H222P mice.

Discussion
The phenotype of a given single-gene mutation in genetically engineered mouse models is modulated by the genetic background. This effect is attributable to modifier genes, which function in combination with the causative gene. Developing congenic mouse strains not only helps us to investigate the effect of LMNA mutations on phenotype but would also facilitate the development of mouse models that more accurately mimic certain features of EDMD. Using validation of single sequence length polymorphism, a method that can be routinely performed, we demonstrated that this approach could be successfully used to certify inbred strains of mouse models of muscular dystrophies. We showed here significant strain-dependent differences in cardiac function between inbred strains of Lmna p.H222P/H222P mice. Particularly, the cardiac phenotype was more pronounced in the 129S2/svPasCrl genetic background. These findings demonstrate the complex effects of genetic divergence between inbred strains on cardiac functions in mouse models of EDMD.
Unlike the C57 Lmna p.H222P mice, we found that 129 Lmna p.H222P mice showed exacerbated arrhythmia susceptibility. This is in agreement with previously reported findings that C57BL/6J mice are resistant to arrhythmia [20,21]. It is known that genetic background dominates the susceptibility to ventricular arrhythmias [22]. In this context, this should be considered when studying cardiac electrical activity in mice. Further exploration of the genetic differences between the two congenic mouse strains, which account for the difference in the cardiac phenotypes of the same Lmna p.H222P mutation, is warranted. Indeed, genomic analysis of the 129 Lmna p.H222P mice and C57 Lmna p.H222P mice will be of interest to identify specific locus associated to arrhythmia. Such a study may provide new mechanistic and clinically relevant insight into the alteration of cardiac function between these strains. Moreover, the relatively sensitive 129S2/svPasCrl mice would be of great value to investigate the genetic basis underlying ventricular arrhythmias. In conclusion the 129 Lmna p.H222P mouse cardiac phenotype was more deleterious than in C57 Lmna p.H222P mouse, at least in part because genetic background of 129S2/svPasCrl present a predisposition for arrhythmia. In human EDMD, arrhythmia-related symptoms frequently preceded heart dilation and heart failure symptoms ( [23]).
In summary, our data suggest that 129 Lmna p.H222P mice have a severe progressive cardiac muscle disease with early-onset deficits. Thus, this mouse model may be a particularly suitable model for evaluating therapeutic strategy for EDMD.

Mouse strains
Lmna H222P/H222P mice [12] were backcrossed eight times with the congenic strains C57BL/6JRj and 129S2/svPasCrl (Janvier Labs). The animals were fed chow and housed in a disease-free barrier facility at

Echocardiography
Mice were anesthetized with 0.75% isoflurane in O 2 and placed on a heating pad (25°C). Echocardiography was performed using an Vivid7 ultrasound with an 11 MHz transducer applied to the chest wall.
Cardiac ventricular dimensions and fractional shortening were measured in 2D mode and M-mode, 3 times per animal. A blinded experimenter, unaware of the genotype performed the examinations.

Electrocardiography
Electrocardiograms were recorded from living mice using the noninvasive ecgTUNNEL ® (Emka Technologies). Waveforms were recorded using Iox Software v1.8.9.18 and intervals were measured with ecgAUTO v1.5.12.50, using the average of three representative consecutive beats. A blinded experimenter, unaware of the genotype performed the examinations.

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 when compared two sets of data, or Kruskal Wallis test with Dunn's test post-test when compared multiple sets data, with a value of P ≤ 0.05 being considered significant. To validate results of echocardiographic analyses, we performed a non-parametric test (Wilcoxon-Mann-Whitney test). Survival curves were generated using the method of Kaplan and Meier, and survival curves comparison was performed with the log rank test (mantel-cox test). Regression analysis was performed for a confidence interval of 95%.