DMD carrier model with mosaic dystrophin expression in the heart reveals complex vulnerability to myocardial injury

Abstract

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease that causes progressive muscle wasting and cardiomyopathy. This X-linked disease results from mutations of the DMD allele on the X-chromosome resulting in the loss of expression of the protein dystrophin. Dystrophin loss causes cellular dysfunction that drives the loss of healthy skeletal muscle and cardiomyocytes. As gene therapy strategies strive toward dystrophin restoration through micro-dystrophin delivery or exon skipping, preclinical models have shown that incomplete restoration in the heart results in heterogeneous dystrophin expression throughout the myocardium. This outcome prompts the question of how much dystrophin restoration is sufficient to rescue the heart from DMD-related pathology. Female DMD carrier hearts can shed light on this question, due to their mosaic cardiac dystrophin expression resulting from random X-inactivation. In this work, a dystrophinopathy carrier mouse model was derived by breeding male or female dystrophin-null mdx mice with a wild type mate. We report that these carrier hearts are significantly susceptible to injury induced by one or multiple high doses of isoproterenol, despite expressing ~57% dystrophin. Importantly, only carrier mice with dystrophic mothers showed mortality after isoproterenol. These findings indicate that dystrophin restoration in approximately half of the heart still allows for marked vulnerability to injury. Additionally, the discovery of divergent stress-induced mortality based on parental origin in mice with equivalent dystrophin expression underscores the need for better understanding of the epigenetic, developmental, and even environmental factors that may modulate vulnerability in the dystrophic heart.

Introduction

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease that involves the heart, resulting in clinically significant cardiomyopathy (1–5). The DMD gene locus on the X-chromosome encodes the protein dystrophin, which plays a critical role in healthy muscle cells by bolstering membrane integrity and serving as a signaling scaffold (6–10). The loss of dystrophin leads to myocyte necrosis, fibrosis, skeletal muscle wasting, and cardiomyopathy, culminating in premature death from cardiorespiratory failure. Due to its X-linked origin, DMD occurs almost exclusively in males at a rate of roughly 1:5000 live births, and in the majority of cases, the disease-causing allele is passed down from a mother that carries one mutated copy of the gene (11–13). Virtually all patients with DMD over 18 years of age display overt clinical signs of cardiomyopathy, including cardiac fibrosis, ventricular dilation, and reduced contractile function (1,14). The devastating cardiac pathology caused by DMD has been recognized since its earliest descriptions, including an 1836 report by Conte and Gioja about two brothers with progressive debilitating muscle weakness, and the oldest of whom evidently suffered from cardiomyopathy at the time of death (1,15). The natural history of the disease often terminates with death from cardiorespiratory failure in the late teens or early 20s. With advancements in symptomatic respiratory therapies and pharmacological management, DMD patient lifespans have been extended into the 30s or early 40s, magnifying the incidence of heart failure in this patient population (16,17).

In recent years, a variety of potential new therapies aiming to enable dystrophin expression in dystrophic muscle have emerged from preclinical work, with a few of them now entering the clinical arena. Some of these therapeutic strategies, like antisense oligonucleotide-mediated exon skipping and CRISPR-Cas9 gene editing, can restore the expression of the endogenous DMD gene for select mutations (18–22). Conversely, micro-dystrophin gene therapy aims to be a universal solution by replacing the endogenous gene with a highly truncated but functional substitute that has been scaled down to its essential components (23–25). These potential therapies hold great promise, and they have all been shown to induce dystrophin expression in both the skeletal muscle and the heart in preclinical studies. However, successful cardiomyocyte transduction continues to be a major obstacle to their cardiac efficiency. In preclinical work, these approaches have often displayed a mosaic pattern of transduction in the heart, with clusters of cardiomyocytes that still lack dystrophin adjacent to those that have been induced to express it (18,21,26–28).

This mosaic dystrophin expression pattern is reminiscent of what is seen in DMD carrier, who harbor one intact copy of the DMD gene and one null allele with a mutation that eliminates expression of the gene. Because of the requirement for two X-chromosomes for carrier status, DMD carriers are genetically female. Due to X-inactivation around the mid-gestational point in embryonic development, female cell nuclei have near-exclusive gene expression from only one of their X-chromosomes (29). Thus, carriers of a null DMD allele might be expected to have dystrophin expression in only half of their myocyte nuclei. Interestingly, skeletal muscles of most DMD carriers are largely protected from injurious effects of dystrophin loss and show a pattern of nearly uniform positive dystrophin expression (30,31). This overwhelmingly positive dystrophin composition may result from selective replacement of dystrophin-negative fibers by dystrophin-positive fibers or may arise from the fact that every skeletal muscle fiber has many independent myonuclei, each with its own X-inactivation status, allowing nuclei producing dystrophin to compensate for those that do not. Skewed X-inactivation is hypothesized to play a key role in the 15% of DMD carriers that do manifest evidence of skeletal muscle damage (29,32), and whose skeletal muscles often display a mixture of dystrophin-positive and dystrophin-negative fibers (33–37). In contrast, the heart displays a mosaic dystrophin expression pattern even in non-manifesting carriers (30,31), reflecting the fact that each cardiomyocyte has only 1–2 nuclei that share the same X-inactivation state. The embryonic timing of X-inactivation and the preservation of this inactivation status during all subsequent cellular and nuclear division events result in the dystrophin expression profile of any given cardiomyocyte being entirely determined by one X-chromosome. Accordingly, cardiomyocytes in the adult heart display clustering in groups that have the X-inactivation status of their shared precursor cell (38,39), giving rise to the mosaic pattern of positive and negative dystrophin expression in DMD carrier hearts (30,31,40).

In animal dystrophinopathy carrier models, the heart has been shown to have roughly 50% dystrophin-positive cardiomyocytes (40). Preclinical gene therapy studies using DMD models show highly variable proportions of dystrophin-positive and dystrophin-negative myocardium after treatment, with efficiency influenced by the type and dose of the therapeutic agent (18,19,26). As these developing therapies move into clinical trials and become available to patients with DMD, it becomes increasingly important to determine how much dystrophin expression is sufficient to rescue the heart in order to establish guidelines for optimal dosing. Dystrophinopathy carrier hearts represent a useful preclinical model to evaluate whether approximately 50% dystrophin-positive myocardium will be sufficient to reduce susceptibility to injury in dystrophic hearts.

Observations in patients with DMD suggest that episodes of elevated cardiac stress and injury may underlie the progressive nature of DMD cardiomyopathy, indicated by transient periods of angina and elevated serum cardiac troponins in the contexts of surgery or other cardiac stress (41–45). We have previously demonstrated that the β-adrenergic receptor agonist isoproterenol (Iso) represents an effective model of episodic cardiac injury in dystrophic mice. Our recent work showed that a single high-dose injection of Iso produced extensive myocardial injury manifesting as widespread clusters of dying myocytes and dramatic elevations in serum cardiac troponins in the mdx mouse heart (46). Previous work by Yue et al. had used low doses of Iso to investigate the impact of transient cardiac stress in mouse DMD carrier hearts (40). The work of Yue et al. showed extensive protection from cardiac stress-induced injury in DMD carrier hearts compared to dystrophin-null mdx female hearts after a brief 12-h course of three low-dose Iso injections. However, our studies fail to reveal a similar degree of cardiac damage after the same low-dose Iso protocol as that reported by Yue et al. Further, after finding a protocol that better matched the previously reported level of mdx cardiac injury, we obtain contrasting results in the dystrophinopathy carrier hearts. In the present report, we demonstrate that DMD carrier mouse hearts with ~57% dystrophin-positive myocardium display significant vulnerability to high-dose Iso-induced myocardial injury and mortality, and that the parental origin of the mutated DMD gene determines the degree of protection afforded by partial dystrophin expression.

Results

DMD carrier cardiac dystrophin expression does not depend on parental origin of the disease allele

Two breeding strategies were used to generate DMD carrier mice, producing maternal (Carrier^M) and paternal (Carrier^P) female DMD carriers from mdx dams and mdx sires, respectively (Fig. 1A). The pattern of dystrophin expression was confirmed to be mosaic, with large adjacent regions of dystrophin-positive and dystrophin-negative cardiomyocytes (Fig. 1B), in agreement with previously published work (40). The presence or absence of dystrophin expression did not affect body weight or heart size at baseline. Body weights at 4–6 months of age were 29.4 ± 0.6, 29.3 ± 0.3, 29.5 ± 0.5 and 31.1 ± 0.6 g for wild type (WT), mdx, Carrier^M, and Carrier^P, respectively. No significant differences were detected in heart weights normalized to tibial length between WT, mdx, and the two groups of carrier female mice (Fig. 1C; P = 0.21).

Figure 1.

Dystrophinopathy carrier hearts display mosaic dystrophin expression throughout the myocardium. (A) Heterozygous DMD carrier mice were generated from two different breeding strategies. Paternal carrier (Carrier^P) mice were bred from mdx sires and wild type (C57Bl/10) dams, and maternal carrier (Carrier^M) mice were derived from mdx dams and wild type sires. (B) Representative whole heart and magnified images of dystrophin distribution in C10, carrier and mdx hearts, showing the mosaicism of carrier dystrophin expression. Magnified image scale = 0.5 × 0.5 mm. (C) Heart weights were not different between C10, carrier and mdx mice at baseline when normalized to tibial length to control for body size (n = 6–14 per group; P = 0.21).

Both breeding strategies resulted in the same degree of dystrophin expression in the carrier hearts, as measured by histological quantification and western blot. Both groups displayed dystrophin-positive signal in approximately 57% of the area corresponding to full dystrophin expression seen in WT hearts, regardless of maternal or paternal origin of the disease-causing allele (56.8 ± 3.7% in Carrier^P and 57.6 ± 3.4% in Carrier^M; Fig. 2A). The magnitude of the range of quantified dystrophin levels in carrier hearts was somewhat surprising, with the lowest-expressing heart having 40.0% dystrophin and the highest-expressing heart having 78.4% dystrophin relative to WT hearts. However, this appears to represent real biological variability, as opposed to an artifact of histological processing (Fig. 2B). The level of dystrophin expression was not litter-dependent, as mice expressing high levels of dystrophin and others expressing lower levels were found in the same litter. The amount of dystrophin expression and the similarity between Carrier^P and Carrier^M hearts were confirmed by western blotting (54.7 ± 3.7% in Carrier^P and 56.1 ± 8.1% in Carrier^M; Fig. 2C and D).

Figure 2.

Dystrophin is expressed in just over half of the heart on average in both groups of dystrophinopathy carriers. (A) Histological quantification of dystrophin-positive signal in DMD carrier hearts normalized to dystrophin signal in wild type (C10) hearts. Both groups of carriers express dystrophin in ~57% of the heart area (n = 7–13 per group). (B) Representative images of biological variability in dystrophin expression in carrier hearts, ranging from 40.0% to 78.4%. (C) Immunoblot quantification of dystrophin content in ventricular tissue, showing ~55–56% average expression of dystrophin in carrier hearts normalized to wild type hearts (n = 3–12 per group). (D) Representative immunoblot bands used for dystrophin quantification.

DMD carrier hearts display intermediate susceptibility to Iso-induced injury that partially depends on parental origin

Previously, Yue et al. showed that 3 IP injections of the β-adrenergic receptor agonist isoproterenol (Iso), each at a dose of 0.35 mg/kg and staggered over a 12-h period before sacrifice, caused necrotic injury in approximately 11% of the mdx heart, but only 2% of the carrier heart, and negligible area in wild type hearts (40). Our attempt to replicate this result with the same Iso dosing protocol yielded significantly lower myocardial damage in mdx and carrier mice. Three sequential Iso injections, at a dose of 0.35 mg/kg each, induced injury in less than 1% of the myocardium in each group, with the mdx injury approximately 2-fold greater than injury in both wild type and carrier hearts (Fig. 3A). The areas of injured myocardium in carrier hearts were largely devoid of dystrophin (Fig. 3B, D and E); however, injured areas in wild type hearts also lost dystrophin staining, suggesting that cardiomyocyte injury likely precipitates dystrophin loss by proteolysis (46).

Figure 3.

Dystrophinopathy carrier cardiac susceptibility is extensive and partially dependent on parental origin of DMD alleles. (A) Three injections of low-dose Iso (0.35 mg/kg each) induced slight injury in C10, carrier and mdx hearts 30 h after the first Iso injection, with 2-fold higher injury area in mdx hearts (n = 6–9 per group; # P = 0.04 vs. mdx). (B) Representative images of hearts 30 h after starting a course of 3 low-dose Iso injections, showing slight IgG-positive (red) cardiomyocyte injury. (C) A single injection of high-dose Iso (10 mg/kg) caused widespread lesions in carrier and mdx hearts and mortality in 21% of Carrier^M mice (n = 17–24 per group; § P = 0.027). Among survivors, both groups of carriers displayed significantly higher injury than C10 hearts, and Carrier^P hearts displayed significantly lower injury than mdx (n = 10–14 per group; ^*P = 0.04 vs. C10; ^***P < 0.001 vs. C10; # P = 0.04 vs. mdx; ## P = 0.001 vs. mdx). (D) Representative images of acute injury distribution in wild type, carrier and mdx hearts after a single high-dose Iso injection. (E) Most of the injured cardiomyocytes (red) lack dystrophin (green), including in wild type hearts; white arrows indicate injured myocytes with significant dystrophin signal. Magnified image scale = 0.3 × 0.3 mm.

In an effort to recapitulate the higher level of injury in mdx hearts presented by Yue et al., our protocol based on a single IP injection of high-dose (10 mg/kg) Iso was used to induce cardiac injury (46). This dose revealed 10-fold higher injury in mdx hearts compared to WT hearts (16.2 ± 1.8% versus 1.8 ± 0.4%, respectively), and variable and intermediate susceptibility to injury in the two groups of dystrophinopathy carriers. Surprisingly, 21% (4 of 19) of Carrier^M mice displayed spontaneous mortality after a single Iso injection, while Carrier^P mice all survived until sacrifice, and only 1 of 24 mdx mice died (Fig. 3C). The mice that succumbed before the predetermined 30-h timepoint after Iso displayed very high IgG-positive myocardial area of 35.1 ± 4.9% in Carrier^M and 36.4% in the single mdx heart, suggesting that the deaths were likely cardiac in nature. However, because it is difficult to determine how much of this damage occurred post-mortem, these mice have been excluded from statistical comparisons based on surviving mice. Surviving carrier mice displayed damaged myocardial area that was significantly higher than in WT hearts and significantly lower than in mdx hearts 30 h after Iso (Fig. 3C). Comparisons of the number of lesions and average lesion sizes between the four groups revealed that these parameters closely mirrored percent lesion area, with similar intra-group variability and no significant differences between the two groups of carrier mice (data not shown). As before, the majority of injured IgG-positive cardiomyocytes lacked dystrophin, although the rare injured myocyte could be found with some dystrophin expression (Fig. 3E).

DMD carriers are highly susceptible to repeated bouts of high-dose Iso but display divergent survival

To determine the extent of protection offered by ~50% dystrophin expression in hearts subjected to repeated injurious stress, mice were subjected to multiple bouts of adrenergic stimulation with high-dose Iso (47). In this protocol, WT, carrier and mdx mice received 14 doses of 10 mg/kg Iso in a span of 5 days before sacrifice (Fig. 4A). As in the case of a single bolus Iso injection, parental origin of the null DMD allele had a significant effect on survival. Among Carrier^M mice, 31% succumbed to the repeated Iso challenge, reflecting nearly identical mortality as mdx mice (38%). However, all Carrier^P mice and WT mice survived the challenge (Fig. 4B).

Figure 4.

DMD carrier hearts are highly susceptible to damage with repeated bouts of injurious stressor. (A) The repeated Iso challenge is comprised of 14 injections of high-dose (10 mg/kg) Iso over the span of 5 days. (B) All of the C10 and Carrier^P mice survived the repeated Iso challenge, but 31% of Carrier^M and 38% of mdx mice died during the challenge (^*P = 0.01; ^**P = 0.005). (C) Among survivors, repeated high-dose Iso caused similarly extensive replacement fibrosis in the hearts of carrier and mdx mice, which was significantly higher than C10 injury (n = 6–17 per group; ^**P = 0.01; ^***P < 0.001). (D) Representative images of fibrosis in wild type (C10), carrier and mdx hearts after repeated injury with high-dose Iso. Fibrosis is red, and intact myocardium is green.

Hearts from mice surviving the challenge were analyzed to determine the area of cardiac fibrosis, a reflection of the amount of myocardium lost during the challenge. Carrier mice were found to have equally high levels of cardiac damage as mdx mice, regardless of maternal or paternal origin of the disease-causing allele (Fig. 4C). Carrier hearts showed a similar distribution of replacement fibrosis as dystrophic hearts, with large lesions scattered randomly throughout the myocardium, including epicardial regions, in contrast to the endocardial localization of WT heart lesions (Fig. 4D).

Discussion

DMD has been described in the literature for nearly 200 years as a devastating, progressive disease that wastes away skeletal muscle and causes heart disease. In the intervening decades, clinical advances that include symptomatic respiratory therapies and scoliosis correction have markedly extended lifespans, but only modest strides have been made in treating DMD cardiomyopathy (16,17). A new wave of emerging gene-targeted therapies holds the promise of potentially correcting the underlying defect in DMD by restoring dystrophin expression, but the efficacy of these approaches in treating the heart remains unknown. A major obstacle to maximizing the cardiac benefits of these corrective strategies lies in differences in tissue transduction between the heart and skeletal muscle that can result in hindered cardiac efficiency. Some preclinical studies of antisense-mediated exon skipping have shown incomplete cardiac dystrophin restoration, leaving a mosaic pattern of dystrophin-positive and dystrophin-negative myocardium (18,19,26). Other studies utilizing AAV gene delivery in large animal models have also resulted in partial restoration of dystrophin expression (21,48). A similar mosaic distribution of dystrophin is seen in the hearts of DMD carriers who have one normal allele and one null allele of the X-chromosomal DMD gene, due to random X-inactivation throughout the myocardium.

In the present study, we have used the mouse model of DMD carriers to determine whether dystrophin expression in approximately half of the heart can protect the remaining myocardium in the context of single or repeated bouts of workload-induced cardiac stress. We report that carrier mice display intermediate susceptibility to a single bout of high-dose Iso-induced injury relative to wild type and dystrophin-null hearts, but they sustain the same degree of injury as dystrophic hearts with repeated injurious Iso stimulation. These findings are also consistent with the clinical picture of DMD carriers who have an increased incidence of cardiomyopathy (32). Importantly, the comparison between carriers derived from two different breeding schemes revealed the surprising relationship between the parent from whom the dystrophin-null allele is inherited and mortality after Iso. Specifically, although all carriers derived from mdx fathers and wild type mothers survived the Iso-induced stress, carrier mice from mdx mothers and wild type fathers showed 21% mortality after a single dose and 31% mortality after repeated doses of Iso, despite identical average dystrophin expression.

The critical implication of these results for DMD therapies is that the degree of protection from mortality in partially dystrophin-expressing hearts likely depends on additional factors besides dystrophin levels. Although average dystrophin levels were very similar in the hearts of both groups of this carrier mouse model, their tolerance of high cardiac workload appears to be altered through a mechanism that significantly affects their vulnerability and survival. Elucidation of this mechanism is beyond the scope of this work; however, differences between in utero environments or epigenetic regulation are likely candidates. The divergence in vulnerability may also involve a more complex basis, like differential psychosocial interactions between healthy and dystrophic mothers with their heterozygous offspring, differences in the timing of X-inactivation, or variable mitochondrial content and bioenergetics. This suggests that in human patients, a variety of factors could influence the degree of therapeutic correction that is sufficient to prevent lethal heart failure, potentially including polymorphisms in other genes, maternal and paternal epigenetic imprinting, and even environmental or dietary factors.

Phenotypic diversity stemming from complex factors besides dystrophin is readily evident in human DMD patients and DMD carriers. Marked phenotypic variability with respect to the degree of cardiac and respiratory disease severity has been documented in multiple sets of brothers diagnosed with the same DMD-causing mutation, without clear evidence of what may cause the divergence (49). Furthermore, the cardiac and respiratory phenotypes in any given patient may progress independently of each other, and with trajectories that are not predictable based on the disease-causing mutation, in a phenomenon referred to as phenotypic discordance (50). Likewise, human carriers of DMD have been documented to have variable skeletal muscle and cardiac phenotypes that were not overtly linked to their underlying mutation (51,52). In fact, the reported incidence of cardiomyopathic symptoms in DMD carriers ranges from <10% to nearly 50%, reflecting the high variability of the carrier cardiac phenotype (51,53). Regardless of the variability in symptomology, current guidelines recommend routine monitoring of carrier cardiac function, reflecting the understanding that DMD carriers are at high risk for developing cardiomyopathy (2,53,54). Together with the preclinical findings presented here, these observations in human patients underscore the notion that the therapeutic efficacy of partial dystrophin restoration in the heart is difficult to predict.

Many of the findings presented here do not align with those reported by Yue et al. in 2004, including the susceptibility to injury observed in carrier mouse hearts and the divergent phenotypes of carriers from the two different breeding strategies (40). Many of these differences may stem from our use of a higher isoproterenol dose, which is the largest deviation in experimental details between previous and current work. In our hands, the low dose of Iso (0.35 mg/kg) previously used in the 2004 study was insufficient to cause more than 1–2% injury in mdx hearts, and thus, we turned to the higher dose of Iso (10 mg/kg) introduced in our previous work to recapitulate more widespread injury in dystrophic hearts (46). The higher dose of this β-adrenergic receptor agonist would be expected to cause more pronounced and sustained increases in heart rate and cardiac contractility, while simultaneously triggering a reduction in blood pressure secondary to peripheral vasodilation. The significant mechanical and metabolic stresses that cardiomyocytes are subjected to during this time are expected to cause more exaggerated myocardial injury after a higher dose of Iso. However, it is unclear what factors underlie the large differences in susceptibility to injury induced by the low dose of Iso between the two studies. While the mice in the present study were 1–3 months older than those used by Yue et al., this is unlikely to account for their increased resilience. Another possible explanation may lie in the use of Evans Blue dye by Yue et al. in contrast to IgG staining used in the current study. Although both measures reflect cardiomyocyte permeability to serum proteins, Evans Blue dye was injected 12 h ahead in the earlier work, and it may have exerted mild cytotoxic effects that were subsequently amplified by Iso-induced cellular stress. A multitude of other unknown factors could also contribute to the overall effect of Iso in conscious animals, including injection timing relative to light/dark cycles, single or group housing, handling during the study, and other environmental influences.

Current evidence points to the strong possibility that the restoration of dystrophin in roughly half of the heart may be sufficient to steer the course of the disease away from heart failure in some patients, but not in others. Further preclinical and clinical research will need to be directed at determining the factors that contribute the overall outcomes in partially dystrophin-replete hearts. Additionally, the hearts of human patients undergoing gene-targeted therapies should continue to be carefully monitored using the latest available guidelines to evaluate the potential need for additional therapeutic interventions with pharmacological therapies like angiotensin receptor blockers or ACE inhibitors.

Materials and Methods

Animals

The wild type control strain C57BL/10SnJ (C10), the dystrophic strain C57BL/10ScSn-Dmd^mdx/J (mdx) and the heterozygous offspring of their crosses (carrier) were bred and maintained at the University of Minnesota. To limit genetic drift, breeding stock have been purchased from Jackson Laboratories every 5–6 generations. All mice were 4–6 months of age at the time of experiments and were housed in static cages with a 12-h light-dark cycle. Since only female animals can be heterozygous with respect to X-chromosome genes like DMD, all mice used in this study were female. All animal procedures were approved by the University of Minnesota Institutional Animal Care and Use Committee and performed in compliance with all relevant laws and regulations. To enable the detection of parentage effects on experimental outcomes, two groups of DMD carrier mice were generated with either maternal (Carrier^M) or paternal (Carrier^P) inheritance of the disease-causing DMD allele. Carrier^M mice were born to mdx dams and C10 sires, and Carrier^P mice were born to C10 dams and mdx sires.

Low-dose isoproterenol challenge

(−)-Isoproterenol hydrochloride (Iso; Sigma #I6504) was dissolved in saline to a concentration of 0.2 mg/ml and sterile filtered into a foil-wrapped glass vial prior to injection. The sterile Iso solution was stored at 4°C for no more than 3 days, and any solution developing discoloration, indicative of degradation, was discarded. Mice received three intraperitoneal bolus injections of 0.35 mg/kg Iso each in volumes of 40–60 μl adjusted for body weight, with 6 h between the first two injections and 3 h between the second and the third injection, as previously described by Yue et al. (40). Mice were sacrificed, and hearts were harvested 30 h after the first Iso injection.

High-dose isoproterenol challenge

Iso was dissolved in sterile saline to a concentration of 6 mg/ml and otherwise handled as described in the section ‘Low-dose isoproterenol challenge’. Mice received a single intraperitoneal bolus injection of 10 mg/kg Iso in volumes of 40–60 μl adjusted for body weight, as previously described (46). Hearts were harvested 30 h after the Iso injection.

Repeated high-dose isoproterenol challenge

Iso was prepared and handled as described in the section ‘High-dose isoproterenol challenge’. Mice received up to 14 injections of 10 mg/kg isoproterenol over the course of 5 days, delivered as 3 daily IP injections at 8 a.m., 12 p.m. and 4 p.m. Hearts from mice that succumbed to the challenge were harvested at the time of discovery, and all survivors were sacrificed at 2 p.m. on the fifth day. Mice that displayed marked and progressive lethargy indicative of a decline toward death were euthanized in compliance with the animal use protocol.

Immunofluorescence and histopathology

At the time of harvest, hearts were cut in half along the short axis, and the apical halves were embedded in OCT blocks for histology. Heart section slides were prepared as previously described (46), and all tissues were unfixed at the time of staining. The following antibodies and reagents were used for immunofluorescence staining: goat serum for blocking (Jackson ImmunoResearch #005-000-121, 10%), rabbit dystrophin monoclonal antibody (Abcam #ab218198, 1:300), goat anti-rabbit IgG (H + L) highly cross-adsorbed Alexa Fluor Plus 647 secondary antibody (Invitrogen #A32733, 1:200), Alexa Fluor 555 goat anti-mouse IgG secondary antibody with minimal cross-reactivity (Biolegend #405324, 1:150), WGA Alexa Fluor 488 conjugate (ThermoFisher #W11261, 5 μg/ml) and ProLong Gold antifade mountant with DAPI (ThermoFisher #P36934). The blocking, primary antibody and secondary antibody incubation steps were all carried out at room temperature for 1 h, separated by three 5-min washes in fresh PBS.

The following reagents were used for Sirius Red Fast Green (SRFG) staining: 1.2% picric acid solution (Ricca #R5860000), Direct Red 80 (Sigma #365548), Fast Green FCF (Sigma #F7252), Formula 83 clearing solvent (CBG Biotech F83) and organic mounting medium (CBG Biotech MM83). Slides were first fixed for 3 h in acetone at −20°C before staining and then rehydrated in 70% ethanol followed by two changes of tap water. The tissue was then stained for 25 min in SRFG dye solution of picric acid, 0.1% Direct Red 80, and 0.1% Fast Green FCF. Staining was followed by three washes in tap water and then dehydration in 70% ethanol, 100% ethanol, and Formula 83 before coverslipping.

All imaging was performed using NIS Elements software on a Nikon Eclipse Ni-E upright epifluorescent microscope with a motorized stage. For dystrophin and IgG lesion analysis, whole heart montages were collected as a stack of four fluorescent channels using a 10x Nikon Plan Apo Lambda objective at a resolution of 0.92 μm/pixel. For evaluation of fibrosis, SRFG-stained sections were imaged as brightfield montages using a 4x Nikon Plan Apo Lambda objective at a resolution of 0.85 μm/pixel.

Histological analysis

Dystrophin was quantified histologically on whole heart montages in Fiji using the threshold function (55). An Alexa Fluor Plus 647 secondary antibody was chosen for dystrophin staining due to strong fluorophore signal and low background signal in the heart in that wavelength range, providing for an excellent signal-to-noise ratio and facilitating this thresholding approach. (Note: the coloration used in the figures represents the choice of optimal colors for depiction rather than the original wavelengths used for imaging). Dystrophin-positive pixel area was divided by total heart section area as measured using the WGA signal. In wild type hearts, this method produced values with a range of 45.4–49.2% with a mean and standard error of 46.9 ± 0.3%, reflecting strong consistency. All dystrophin measurements from individual hearts were then normalized to this wild type dystrophin mean value to determine the relative percentage of dystrophin expression in each heart compared to wild type hearts. The small number of revertant dystrophin-positive myocytes present in all mdx hearts enabled the selection of an appropriate threshold for representing positive dystrophin signal in overwhelmingly dystrophin-negative images.

IgG-positive injury area was determined using whole heart montages in Fiji by subtracting the WGA channel (staining extracellular matrix) from the IgG channel to better distinguish intra-myocyte IgG signal. The resulting IgG image was then thresholded for positive signal to measure total lesion area and normalized to total heart section area.

Fibrosis was analyzed in whole heart montages of SRFG-stained sections using the color threshold function in Fiji. Areas containing Sirius Red-stained collagen were quantified by measuring the pixel area corresponding to a red hue above a minimum saturation threshold. Total heart section area was represented by the full range of hues above the same minimum saturation threshold, and fibrosis was calculated as the Sirius Red area normalized to total heart area. Only hearts from mice that survived the repeated Iso challenge were used for fibrosis quantification following the challenge. Hearts that succumbed earlier in the challenge displayed a complex mix of degenerating myocytes with acute IgG-positive injury and the fibrosis that was in the process of replacing them and were thus difficult to quantify for total damaged area.

Western blot

Unfixed heart cryosections, cut to seven microns and stored on slides in the −80°C freezer, were used for protein extraction. Six heart sections per mouse were rehydrated in PBS to remove OCT and then scraped with a blade from the surface of the slide into a tube. The tissue was homogenized and allowed to incubate in sample buffer (325 mm Tris HCl, 575 mm sucrose, 5% beta-mercaptoethanol, 15% SDS) for 10 min at 37°C. The samples were then gently homogenized a second time and loaded into a 4–20% acrylamide gradient gel. The gel was run at 75 mA for 1 h. After the separation, proteins were electrophoretically transferred onto a PDVF membrane using a constant current of 250 mA at 4°C overnight. Confirmation of successful protein transfer was performed by Ponceau S staining (Sigma P3504).

The following reagents were used for immunoblotting of the membrane: 5% milk for blocking, rabbit dystrophin polyclonal primary antibody (Abcam ab15277, 1:2000), mouse actin monoclonal antibody (DSHB JLA20, 1:2000), Alexa Fluor 680 goat anti-rabbit IgG secondary antibody (Invitrogen A21058, 1:2000) and Alexa Flour 680 goat anti-mouse IgM secondary antibody (Invitrogen A21048, 1:2000). The reagents were diluted in PBS + 0.05% tween 20. The blocking, primary antibody and secondary antibody steps were 1 h each, separated by 5-min washes in PBS + 0.05% tween 20. The membrane was imaged on the Odyssey CLx (LiCor) at 680 nm excitation.

Statistics

All statistical analyses were performed using Prism 7 (GraphPad Software). Dystrophin and injury levels were compared across groups using one-way ANOVA with Holm-Sidak post hoc test. Survival after a single high-dose Iso injection was compared using the Chi-square test, and survival over time in the repeated Iso challenge was compared using a log-rank test with the Bonferroni correction to adjust for multiple comparisons. All descriptive statistics and columns in graphs reflect the mean ± standard error.

Funding

National Institutes of Health (R01 HL114832 and K08HL102066 to D.T., F31HL139093 to T.A.M.); Muscular Dystrophy Association (351960 to D.T.).

Conflict of Interest Statement: The authors have no conflicts of interest to report.