Evans blue dye uptake assay for membrane leak

Evans blue dye (Sigma, E-2129) was performed as described [19,21]. Evans blue dye (Sigma, E-2129) was dissolved in phosphate-buffered saline at 10 mg/ml and injected intraperitoneally at 5 μl/g body weight. Twenty to 40 h later, the tissues were harvested, finely minced, weighed and incubated at 55°C in 1 ml formamide for 2 h before spectrophotometric absorbance was measured at 620 nm [22,23]. Results are reported as absorbance/mg tissue.

Hydroxyproline assay for fibrosis

The hydroxyproline (HOP) assay was performed as described [19,21,24]. The tissue was minced, weighed and hydrolyzed overnight in 2 ml of 6 M hydrochloric acid at 110°C. Ten μl of this hydrolysate was mixed with 150 μl isopropanol, then 75 μl of 1.4% chloramine-T (Sigma, St Louis, MO) in citrate buffer and oxidized at room temperature for 10 min. One ml of a 3:13 solution of Ehrlich’s reagent (3 g of 4-(dimethylamino) benzaldehyde, Sigma, St Louis, MO; 10 ml ethanol; 675 μl sulfuric acid) to isopropanol was added, mixed and incubated for 30 min at 55°C followed by extinction measurement at 558 nm. A standard curve (0–5000 nM, trans-4-hydroxy-L-proline, Sigma, St Louis, MO) was included in each assay. Results are reported as nM HOP/mg tissue.

Immunofluorescence microscopy

Tissues were flash frozen in liquid nitrogen-cooled isopentane and stored at 80°C; 7-μm sections were cut on a cryostat and fixed to slides in ice-cold 100% methanol. The following antibodies were used: dystrophin NCL-DYS2 (Novocastra/Leica), PH3 04–817 (Millipore), CD3 MON1003-1 (Monsanto), caspase 3 (BD Biosciences), MAC1 BD557395 (BD Biosciences) and eMHC F1.652 (ATCC). The TUNEL kit was from Millipore. Central nuclei and fiber size variability was determined blinded to genotype by analyzing ten randomly chosen fields of 40× magnification. Fiber size variability was compared using each animal’s coefficient of variability (standard deviation/mean).

Echocardiography

Twelve-week animals were evaluated by echocardiography as described [25,26]. Investigators were blinded to genotype. To avoid the stress associated with conscious restraint, anesthetized animals were studied. Anesthesia was induced by 1% isoflurane in a closed chamber (Ohmeda Fluotec 3; Matrix Medical, Orchard Park, NY) in 20% O₂ delivered through a nose cone. Chest hairs were removed with a topical depilatory agent. Limb leads were attached for electrocardiogram gating, and the animals were imaged in the left lateral decubitus position with a Visual Sonics Vevo 770 machine using a 30-MHz high-frequency transducer. Body temperature was maintained using a heated imaging platform and warming lamps. Anesthesia was variably delivered to maintain heart rates throughout the procedure at a constant 380–420 beats per minute. Two-dimensional images were recorded in parasternal long- and short-axis projections, with guided M-mode recordings at the midventricular level in both views. LV cavity size and wall thickness were measured in at least three beats from each projection and averaged. LV wall thickness, interventricular septum (IVS) and posterior wall (PW) thickness, and internal dimensions at diastole and systole (LVIDd and LVIDs, respectively) were measured. LV fractional shortening [(LVIDd – LVIDs)/LVIDd] and relative wall thickness [(IVS thickness + PW thickness)/LVIDd] were calculated from the M-mode measurements.

The MRL background does not protect against membrane leak

Disruption of the membrane-associated dystrophin complex renders the sarcolemma unusually fragile, leading to abnormal membrane leakage that is visualized by uptake of the nonspecific vital tracer Evans blue dye [20]. Upon gross inspection, the muscles from the Sgcg^MRL/D2 mice displayed high levels of dye uptake, comparable to what was observed in the parental Sgcg mice (Additional file 1: Figure S1). On a microscopic level, dye-positive cells were readily detectable in Sgcg^MRL/D2 muscle and heart (Figure ​(Figure4).4). Evans blue dye levels were measured in multiple muscle groups and were not significantly different between Sgcg^MRL/D2 and Sgcg^D2 muscles for quadriceps, triceps, gastrocnemius/soleus, gluteus and abdominals muscle groups (Figure ​(Figure5).5). We also measured dye uptake in older animals at 32 weeks to assess disease progression. At 32 weeks, the skeletal muscles continued to show comparable levels of membrane leak as what was seen at 8 weeks (Figure ​(Figure5).5). The gluteus/hamstring group of muscles showed an increased of dye uptake compared to the 8-week animals. This increase was not seen for other muscle groups.

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Figure 4

Membrane leak is not corrected by the presence of the MRL genome in Sgcg heart and muscle. Evans blue dye is found in cardiomyocytes and skeletal myofibers reflecting membrane leakiness. Dye uptake occurs in patchy pattern throughout the heart and muscle and is seen as opacified cells that fluoresce red. Nuclei are shown in blue and dystrophin in green.

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Figure 5

The MRL genome does not significantly reduce membrane damage in Sgcg muscle. Evans blue dye uptake is a measure of membrane damage and is represented on the y axis (absorbance/mg). In the quadriceps, triceps, abdominal and gastrocnemius/soleus muscles, membrane damage is not significantly different between Sgcg^D2 animals and intercrossed Sgcg^MRL/D2 animals at both the 8- and 32-week time points. In the gluteus/hamstring muscles, membrane damage is significantly (**p < 0.01) increased at the 32-week time point in intercrossed Sgcg^MRL/D2 animals compared to the 8-week time point.

We compared suppression of dye uptake by the MRL background to that induced by the 129T2/SvEmsJ background in Sgcg mice (Figure ​(Figure6).6). The quadriceps and abdominal muscles from Sgcg null mice with a contribution from the 129T2/SvEmsJ background showed increased membrane leak compared to Sgcg mice with an MRL contribution. However, the triceps muscle group from the MRL background showed increased dye uptake, and the gluteus/hamstring muscle group showed no significant difference. Thus, there was no consistent suppression of membrane leakiness by the MRL strain.

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Figure 6

The MRL genome has a variable ability to reduce membrane leak in muscular dystrophy compared to the 129T2/SvEmsJ strain. Evans blue dye uptake was measured in multiple muscle groups represented on the y axis (absorbance/mg). Membrane damage is reduced in the quadriceps (**p = 0.0053) and abdominal (**p = 0.0012) muscles of intercrossed Sgcg^D2/MRL animals compared to intercrossed Sgcg^D2/129 mice. Membrane damage is increased in the triceps muscle (*p = 0.0298) of intercrossed Sgcg^MRL/D2 animals. There is no difference in membrane damage in the gluteus/hamstring muscle.

The MRL genome may promote skeletal muscle regeneration

The MRL background is thought to exert part of its effect by promoting regeneration [34]. We examined embryonic myosin heavy chain (eMHC) expression as a reflection of regeneration. Sgcg^MRL/D2 muscle shows patchy areas with a qualitative increase in eMHC-positive fibers compared to Sgcg (Figure ​(Figure7),7), and wild-type muscle showed no eMHC positive fibers (data not shown). In dystrophic skeletal muscle, ongoing regeneration is thought to offset degeneration. Consistent with this, eMHC-positive regions were also positive for Evans blue dye uptake, indicative of muscle damage. We also evaluated phosphorylated histone 3 (PH3) as a reflection of mitotic index. No clear differences for PH3 staining were seen between Sgcg^MRL/D2 and Sgcg muscle. Regenerating skeletal muscle, whether from trauma or muscular dystrophy, is identified by the presence of myofibers with centrally positioned nuclei. In muscular dystrophy, ongoing regeneration is also marked by increased fiber size variability. Sgcg^MRL/D2 mice have an increased number of central nuclei in quadriceps and diaphragm muscle compared to Sgcg^D2 mice (Figure ​(Figure7).7). These same muscles also showed increased fiber size variability when there is a contribution from the MRL background. These data are consistent with a model in which favorable matrix remodeling may support enhanced regeneration.

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Figure 7

Regeneration may be enhanced in the MRL background. Embryonic myosin heavy chain (eMHC) staining was increased in Sgcg^MRL/D2 compared to Sgcg muscle, and eMHC fibers were seen in areas where dye uptake was seen. However, phosphorylated histone 3 (PH3), a marker of mitotic index, was not increased in Sgcg^MRL/D2 compared to Sgcg muscle. Skeletal muscle central nucleation and fiber size variability are enhanced by the MRL background. Sgcg^MRL/D2 muscle demonstrated an increase in centrally nucleated fibers compared to Sgcg^D2 mice. Fiber size variability was reflected in the coefficient of variability of fiber diameter measurements. The solid line indicates significance (p < 0.05). These data support that the MRL exerts its effect in skeletal muscle, at least in part, by promoting regeneration.

We also assessed apoptosis using a TUNEL assay and caspase 3 staining and again found no differences between Sgcg^MRL/D2 and Sgcg muscle, suggesting that gross differences in programmed cell death are unlikely to account for the improved healing of the MRL strain (Additional file 1: Figure S2). We also characterized whether T cell infiltration or macrophage infiltration differed qualitatively between Sgcg^MRL/D2 and Sgcg muscle, but we found no clear differences between Sgcg^MRL/D2 and Sgcg muscle, suggesting other features contribute to the MRL background’s effect on muscular dystrophy (Additional file 1: Figure S2).

Improved cardiac function from a 50% contribution of the MRL background

We performed 2D and M mode echocardiography on Sgcg^MRL/D2 and Sgcg^D2 mice at 12 weeks of age. Both fractional shortening and the left ventricular ejection fraction were significantly reduced in Sgcg^D2 mice, indicating that the increased fibrosis impairs cardiac function (Table ​(Table2).2). Fractional shortening and the left ventricular ejection fraction were similar between Sgcg^MRL/D2 and WT^MRL/D2 mice, consistent with improved function mediated by 50% of the MRL genome. Wall thickness was also increased in Sgcg mice compared to the wild type of the same background. In contrast, wall thickness was not different between Sgcg^MRL/D2 and WT^MRL/D2 mice.

Chromosome 2 associates with reduced fibrosis Sgcg heart and diaphragm muscle

We conducted a genome-wide scan using markers that were informative in the two parental strains DBA/2J and MRL/MpJ (Figure ​(Figure8).8). QTLRel was used to identify regions of association; this analysis takes into account the relatedness of individual animals in the cohort [29,30]. A region on chromosome 2 was identified that associated with fibrosis in the heart. The 1.5-LOD drop interval of this region spans from 64.8008−73.1758 cM in mouse genome build 37.1. This same region was also associated with fibrosis in the diaphragm muscle and also for fibrosis in the abdominal muscles. It should be noted that the significance of these associations is suggestive (p < 0.63) when using the stringent QTLRel analysis. However, the overlapping intervals found in heart, diaphragm and abdominal muscles provide additional support that this interval modifies fibrosis.

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Figure 8

Fibrosis in the heart, diaphragm and abdominal muscles is modified by a locus on chromosome 2. Genome-wide association was examined for fibrosis in Sgcg^MRL/D2 progeny in the (A) cardiac muscle (n = 65), (B) diaphragm (n = 78) and (C) abdominal muscle (n = 78). Chromosomes are plotted on the x axis, and the informative SNPs tested (n = 1,707) are displayed as open circles that alternate color by chromosome; LOD scores are represented on the y axis. Overlapping regions on chromosome 2 showed suggestive (p < 0.63) association with fibrosis in heart, diaphragm and abdominal muscle.

Chromosome 2 candidate genes

When considering the overlapping region on chromosome 2 from the heart, diaphragm and abdominal fibrosis data, the interval contains 49 known genes. There are additional predicted and unnamed genes and genes encoding olfactory receptors that are not considered likely candidates. Within the interval is Jag1, encoding jagged the ligand for the Notch receptor. Loss of function of Notch 3 leads to muscle hyperplasia, especially when subjected to repetitive injury [38]. Ex vivo activation of Notch signaling helps maintain donor cell engraftment during myoblast transfer [39]. Another gene linked to growth and healing in the interval is Bmp2-encoding bone morphogenetic protein 2. The BMPs mediate musculoskeletal regeneration [40], and given the MRL background effect on multiple cells and tissues, BMP2 is well positioned to contribute to the MRL superhealing response. That said, this chromosome 2 interval has not previously been linked to other MRL healing properties. The strict criteria imposed by the QTLRel algorithm makes it unlikely that these results derive from relatedness of animals within the cohort.

Cardiac injury and repair in the MRL strain

The ability of the heart to recover after injury has been studied in the MRL strain using cryoinjury, left anterior descending ligation and ischemia reperfusion methods [5,6,8-10,14,15]. However, scar reduction has been noted after some forms of injury while not after others. In common to all these studies is an acute injury model where a normal heart was substantially damaged in a single setting. The MRL’s ability to heal the heart may be limited such that larger amounts of injury may be insurmountable, as proposed by Naseem [6]. Our data support that lower, although persistent, levels of injury can be managed by the MRL strain where there is significant reduction in fibrosis and a corresponding functional improvement in cardiac function.