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. Author manuscript; available in PMC: 2015 Feb 26.
Published in final edited form as: Exp Gerontol. 2013 Nov 12;49:26–34. doi: 10.1016/j.exger.2013.10.015

Mechanical and non-mechanical functions of Dystrophin can prevent cardiac abnormalities in Drosophila

Ouarda Taghli-Lamallem a,b,*, Krzysztof Jagla b, Jeffrey S Chamberlain c, Rolf Bodmer a,**
PMCID: PMC4340692  NIHMSID: NIHMS655661  PMID: 24231130

Abstract

Dystrophin-deficiency causes cardiomyopathies and shortens the life expectancy of Duchenne and Becker muscular dystrophy patients. Restoring Dystrophin expression in the heart by gene transfer is a promising avenue to explore as a therapy. Truncated Dystrophin gene constructs have been engineered and shown to alleviate dystrophic skeletal muscle disease, but their potential in preventing the development of cardiomyopathy is not fully understood. In the present study, we found that either the mechanical or the signaling functions of Dystrophin were able to reduce the dilated heart phenotype of Dystrophin mutants in a Drosophila model. Our data suggest that Dystrophin retains some function in fly cardiomyocytes in the absence of a predicted mechanical link to the cytoskeleton. Interestingly, cardiac-specific manipulation of nitric oxide synthase expression also modulates cardiac function, which can in part be reversed by loss of Dystrophin function, further implying a signaling role of Dystrophin in the heart. These findings suggest that the signaling functions of Dystrophin protein are able to ameliorate the dilated cardiomyopathy, and thus might help to improve heart muscle function in micro-Dystrophin-based gene therapy approaches.

Keywords: Micro-Dystrophins, Heart period, Myofibrils, Nitric oxide synthase, Aging

1. Introduction

Dystrophinopathies are due to mutations in the Dystrophin (Dys) gene causing Duchenne and Becker muscular dystrophy (DMD and BMD, respectively) and X-linked dilated cardiomyopathy. In all these pathologies, the heart muscle is affected to different degrees, depending on the type of the mutation and the progression of the disease. Cardiac disease in both DMD and BMD manifests itself as a dilated cardiomyopathy (DCM) and/or cardiac arrhythmia (Corrado et al., 2002). Cardiomyopathy is present in about 90% of DMD/BMD patients, and progressively leads to heart failure, causing the death of 20% DMD and 50% BMD patients (Bushby et al., 2003).

Currently, there are no effective treatments of DCM besides transplantation and pharmacological intervention, such as with angiotensin-converting enzyme inhibitors and beta blockers, which only ameliorate heart symptoms, without correcting the underlying pathology (Kaspar et al., 2009). Inevitably, the DCM worsens as the patient becomes older and as the disease progresses. New therapy alternatives to manage the DCM are thus needed. Viral-based gene therapies, including the adeno-associated virus (AAV) system have recently drawn considerable attention for exploring the potential utility of a modified, but functionally active Dys gene in ameliorating the dystrophic skeletal and heart muscle phenotypes (Bostick et al., 2011; Gregorevic et al., 2006).

Two essential functions have been attributed to Dys protein. The first one is referred to as a mechanical role: Dys links to F-actin via its N-terminal and central actin-binding domains, and to Dystroglycan (Dg) via its WW and cysteine-rich (CR) domains, thus enabling force transduction from the inside to the outside of the cell, and stabilizing the sarcolemma. The second is a signaling role: Dys assembles signaling molecules, including neuronal nitric oxide synthase (nNos), growth factor receptor-bound protein 2 (Grb2), Calmodulin, and Calmodulin-dependent kinases (Anderson et al., 1996; Brenman et al., 1996).

Previous work in skeletal muscle of the mouse DMD model (“mdx”) showed that expressing Dys isoforms preserving its mechanical function is beneficial, by improving mdx muscle function and preventing dystrophy (Gregorevic et al., 2006; Harper et al., 2002). Other studies suggest that restoring the Dys-glycoprotein complex (DGC) by expressing the Dp71 or the Dp116 isoforms in skeletal muscle of mdx mice, which lack the mechanical function of Dys, does not ameliorate the dystrophic phenotypes (Greenberg et al., 1994; Rafael et al., 1996). These data demonstrate that the mechanical role of Dys protein is the major contributor to the mdx dystrophic pathology. However, Dp116 expression in mouse mutants lacking both Dys and Utrophin (mdx:utrn−/−) increases the muscle mass and improves growth, mobility and lifespan (Judge et al., 2011). Dp116 is a non-muscle isoform specifically expressed in Schwann cells of the peripheral nervous system that binds to the DGC components Dg, syntrophin (Syn) and dystrobrevin (Dbr), but does not provide a link to F-actin (it lacks the actin-binding domains). Thus Dp116 allows the assessment of the functional characteristics of the Dys ‘signaling domain’ in the absence of the ‘mechanical domain’ function.

While many studies have focused on structural–functional analysis of Dys in skeletal muscle to develop gene therapy [reviewed by (Blankinship et al., 2006)], little effort has so far been directed to correcting the heart pathology (Bostick et al., 2009, 2011; Hainsey et al., 2003; Townsend et al., 2007; Yue et al., 2003). To differentiate between the ‘mechanical’ and ‘signaling’ roles of Dys in cardiac muscle function, we generated flies that express either the Dys constructs ΔH2-R19/ΔCT and ΔR4-23/ΔCT, which can bind the F-actin, but lack the C-terminal domain that interacts with Syn and Dbr (predicted Dys with ‘mechanical function’) or the Dp116 with the C-terminal domains only, thus lacking the F-actin-binding domains (predicted Dys with ‘signaling function’). We note that probably none of these Dys constructs are involved in nNos signaling, since they lack repeat 16 and 17 of the rod domain implicated in the interaction between nNos and Dys (Lai et al., 2009). We provide evidence that both the predicted ‘mechanical’ (ΔH2-R19/ΔCT, ΔR4-23/ΔCT) and ‘signaling’ (Dp116) functions of Dys are able to ameliorate dilated cardiomyopathy and improve myofibrillar organization. Manipulating Dp116 and Nos in Dys−/− mutants provides further evidence of a signaling role of Dys in modulating the heart function. We conclude that both ‘mechanical’ and ‘signaling’ roles of Dys are important for cardiac muscle function.

2. Materials and methods

2.1. Drosophila strains

The flies Dys−/− are transheterozygous for DysExel6184 and Df(3R) Dl-X43 (Taghli-Lamallem et al., 2008). The DysEP(3)3397 and the Dystroglycan mutants were a generous gift from R. Ray. GAL4 drivers were: 24B-GAL4 (Brand and Perrimon, 1993) and Hand-Gal4 (from A. Paululat laboratory) kindly offered by L. Perrin. UAS-NosRNAi from VDRC (transformant ID 27725) and the UAS-Nos flies were generously offered by S.A. Davies and P.H. O’Farrell.

2.2. Dystrophin transgenic flies

The murine Dys cDNAs ΔH2-R19ΔCT, ΔR4-23ΔCT and Dp116 have already been described (Harper et al., 2002; Judge et al., 2006). The ΔH2-R19ΔCT and ΔR4-23ΔCT lack a portion of the rod domains (spectrin repeats) and the C-terminal region of Dys. Dp116 is a short C-terminal isoform, containing two and a half spectrin repeats, the WW domain, the cysteine-rich domain, and the carboxy-terminal domain involved in binding to other DGC proteins like Syn and Dbr (for details of the construct, see Judge et al., 2006). For the generation of transgenic flies, ΔH2-R19ΔCT; ΔR4-23ΔCT; and Dp116 cDNA constructs were sub-cloned into the Gal4-inducible vector pUAST at the NotI site, injected into w1118 embryos, and transgenic lines established. The transgenic flies were crossed to the heterozygous deficient flies Df(3R) Dl-X43 to generate a stock UAS-ΔH2-R19ΔCT (or UAS-ΔR4-23ΔCT or Dp116)/cyo, Df(3R)Dl-X43/TM3. These lines were crossed to Hand-Gal4, DysExel6184/TM3 flies to generate the transgenic rescue flies with the truncated Dys.

2.3. Immunostaining of adult Drosophila hearts

Staining of adult flies was performed as described previously (Taghli-Lamallem et al., 2008). Primary antibodies: rabbit anti-Dystroglycan diluted at 1/1000 (gift from A. Wodarz); phalloidin-cy3 diluted at 1/1000, and two rabbit NOS antibodies diluted at 1/400 (gift from P.H. O’Farrell) and 1/100 (Thermo-Scientific, PA1-039). Secondary antibodies: donkey anti-rabbit conjugated to CY3 (Jackson ImmunoResearch) at 1/300. Immunostained preparations were visualized on Olympus FV300 or Zeiss LSM 510 laser scanning confocal microscopes.

2.4. Heart physiological analysis

Flies anesthetized with fly nap (Carolina Biol., Corp.) were aligned on a dish (dorsal down) and dissected to expose the heart for optical recording by previously described protocols (Fink et al., 2009; Ocorr et al., 2007a). Beating heart images were acquired at rate of about 130 frames per second using Simple PCI software (Compix, Sewickley, PA). Cardiac parameter measurements were quantified and generated using the MatLab-based image analysis program (Fink et al., 2009). M-modes illustrate movements of the heart tube edges in the y-axis over time in the x-axis, generated by excising and aligning a single pixel-wide image from successive frames. Heart periods (HPs) are defined as the time between the ends of two consecutive diastolic intervals. Single HPs were plotted in the histograms to see overall distribution and clustering of HPs. We used Prism software, one-way ANOVA analysis and a Tukey test to process statistics on 20 flies for each genotype.

3. Results

3.1. Dystrophin proteins carrying either the ‘mechanical’ or ‘signaling’ domains ameliorate Dystrophin-deficient heart dysfunction

The large size of the Dys transcript (14 Kb) presents a major challenge for successful gene transfer with viral vectors. This limitation has led to the construction of mini- and micro-Dys genes (Scott et al., 2002). Among these are the micro-Dys constructs ΔH2-R19/ΔCT and ΔR4-23/ΔCT, both maintaining the N-terminal, some of the rod, and cysteine-rich domains, but lacking the C-terminal domain that directly binds to Dbr and Syn, also components of the DGC. These truncated proteins are therefore expected to retain the capacity of force transduction in the sarcolemma, but they may have some ‘signaling function’ (Fig. 1A) (Scott et al., 2002). By contrast, Dp116 Dys contains intact CR and carboxy-terminal (CT) domains, but it lacks the N-terminal and most of the rod domains, preventing its link with the actin cytoskeleton and the mechanical reinforcement at the sarcolemma (Fig. 1A) (Judge et al., 2006).

Fig. 1.

Fig. 1

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Dystrophin (Dys) mechanical and signaling functions prevent dilated cardiomyopathy. (A) Scheme illustrating the structure of Dys and the truncated proteins tested in Dys−/− flies. N: NH2-terminal actin-binding domain (ABD1); H: hinge; R: spectrin-like repeat; actin-binding domain 2 (ABD2); CR: cysteine-rich domain; CT: carboxy-terminal domain; Dg: Dystroglycan; Dbr: dystrobrevin; Syn: syntrophin. Domains in white are deleted domains. The ΔH2-R19ΔCT and ΔR4-23ΔCT lack the CT and are deleted from the rod domain hinge 2 to repeat 19 and repeat 4 to 23, respectively. Dp116 lacks the actin-binding domains and retains only two repeats (R23-R24) with intact CR and CT domains (Judge et al., 2006). (B) Heart systolic diameters for 1- and 5-week-old flies. (C) Percent fractional shortening (%FS) represents an estimation of the heart tube contractility. Significant differences were determined by one-way ANOVA (*p < 0.05; **p < 0.005; ***p < 0.0005). Error bars indicate SEM. N = 20 flies for each genotype. (D) Representative M-mode traces (5 s) illustrating movements of heart tube walls (y-axis) over time (x-axis). Diastolic (black) and systolic (white) diameters were indicated in each M-mode trace. (E) Representative confocal images (stacks) of adult hearts (A3 segment) stained with phalloidin, showing myofibrillar organization. The sarcomeric units are oriented longitudinally as indicated by arrows, and are seen in 57% of Dys−/−; Hand-Gal4 > ΔH2-R19ΔCT, in 71% of Dys−/−; Hand-Gal4 > ΔR4-23ΔCT and in 50% of Dys−/−; Hand-Gal4 N Dp116. Ostia in the myocardium are inflow tracks indicated by arrowheads. Scale bar 10 μm.

We had previously found that the heart of Dys−/− mutant flies (Df(3R)Dl-X43/DysExel6184) was dilated and exhibited abnormal heart performance and contractility, reminiscent of dilated cardiomyopathy in mammals (Taghli-Lamallem et al., 2008). To probe potential differences between Dys mechanical and signaling roles, we generated transgenic flies that express Dys constructs ΔH2-R19/ΔCT, ΔR4-23/ΔCT, or Dp116 in all muscles (24B-Gal4 driver) or specifically in the heart (Hand-Gal4 driver). Our choice of constructs was made to differentiate between portions of Dys that may or may not confer the mechanical reinforcement at the cell membrane, based on mammal studies in skeletal muscles. First, we tested the heart- and muscle-specific effects of these micro-Dys constructs in a wildtype background, and found that their overexpression in mesodermal tissues does not induce cardiac abnormalities (Fig. S1). We then expressed ΔH2-R19/ΔCT, ΔR4-23/ΔCT, or Dp116 in all muscles or heart alone, to attempt to restore heart function in Dys−/− flies. Cardiac physiology was assessed by analyzing high-speed optical recordings of beating hearts in young and old flies (1 and 5 weeks old) (Fink et al., 2009). Expression of the truncated Dys constructs in Dys−/− caused a significant decrease in systolic diameters, and a corresponding increase in fractional shortening (Fig. 1B, C). This suggests a robust rescue of systolic function and heart contractility by both ‘mechanical’ and ‘signaling’ Dys constructs. We note that the pan-mesodermal driver 24B-GAL4 showed a stronger effect than the Hand-GAL4. This difference could arise because the 24B-GAL4 expression in the ventral muscle layer containing the longitudinal myofibrils (skeletal muscle-like associated with cardiomyocytes) could influence the heart function. M-mode traces of 5-week-old Dys−/− and rescue hearts illustrate the dynamics of heart wall movements (Fig. 1D).

Dys−/− mutant hearts exhibit progressively uncompact and disorganized transverse myofibrils with age (Fig. 1E) (Taghli-Lamallem et al., 2008). Examining whether expression of Dys constructs can restore the cytoarchitecture of Dys−/− cardiomyocytes, we found that compared with Dys−/− mutant alone, hearts expressing the Dys constructs displayed a more compact myofibrillar arrangement as revealed with actin–phalloidin staining (Fig. 1E). Importantly, we observed that in some rescued cardiomyocytes the array of the sarcomeric units was oriented longitudinally (Fig. 1E, arrows indicate orientation of nearby myofibrils), indicating that micro-Dys and Dp116 proteins do not have full capacity to restore normal myofibril orientation. Together, these results suggest that like the micro-Dys, Dp116 is able to restore myofibrillar integrity and contractility.

3.2. Dystroglycan does not play a critical role in Drosophila heart function

It is unclear whether in heart tissue the integrity of the Dg–Dys interaction is critical for heart function. This prompted us to determine Dg localization in Dys-deficient hearts, in Dys−/− hearts expressing the truncated Dys constructs, and whether Dg null mutants showed heart phenotypes similar to Dys−/−. Similar to Dys, Dg protein delineates the plasma membranes of the cardiomyocytes (Fig. 2A, B). Dys−/− mutant hearts show a moderately reduced amount of Dg protein (Fig. 2C). Expression of Dys constructs in Dys−/− mutant hearts does not seem to appreciably restore Dg protein (Fig. 2D; Fig. S2), consistent with the idea that rescue of Dys−/− mutant hearts by micro-Dys constructs is not achieved by increasing sarcolemma Dg localization.

Fig. 2.

Fig. 2

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Dystroglycan (Dg) null mutants do not affect heart function. (A–D) Representative confocal stacks of adult hearts stained with Dg antibody. Dg is found at the cell membrane of the cardiomyocytes. Arrowheads indicate Dg localization. (E–G) Bar graph representations of changes in heart chamber dimensions. Note that Dg null mutants do not show decreased fractional shortening. N = 20–30 flies per genotype. One-way ANOVA analysis was used for statistics and p values <0.05 were considered significant (*p < 0.05; **p < 0.005; ***p < 0.0005). Error bars indicate SEM.

To address this further, we characterized the fly heart’s contractile properties of Dg null mutants (Dg055/Dg086: Dg−/−) (Christoforou et al., 2008). We found that Dg−/− flies and double heterozygotes for Dys and Dg did not exhibit a dilated heart phenotype nor reduced fractional shortening (Fig. 2E–G), thus preserving systolic function compared with Dys mutants (Dys−/− and DysEP3397/DysExel6184). Conversely, Dg overexpression in cardiomyocytes caused an increase in systolic and diastolic diameters without significantly affecting the fractional shortening (Fig. 2E–G), suggesting that cardiac contractility was not compromised. Taken together, these results suggest that Dg is not required for Drosophila adult heart function.

3.3. Dp116 Dystrophin increases the heart period in Dystrophin mutant flies

We showed previously that Dys−/− mutants display an increase in heart rate with age (Taghli-Lamallem et al., 2008). To investigate the effects of truncated Dys constructs on heartbeat length, we characterized the cardiac wall dynamics by measuring the time intervals of systolic and diastolic phases at 1 and 5 weeks of age. The mean HPs of wildtype and of Dys−/− both increased with age, but less so in Dys−/− flies (Fig. 3A). Cardiac-driven Dp116 expression in Dys−/− mutants, however, resulted in significantly prolonged HPs compared with Dys−/− flies at both ages (Fig. 3A), mainly due to increased diastolic intervals (Fig. 3B, C). The HP distribution is represented in histograms, showing a tight clustering at a young age, which expands because of increased age-dependent arrhythmias, but does not significantly change with genotype (Fig. 3D). This suggests that the C-terminal domain of Dys likely plays a role in regulating heart rate, in addition to contractility.

Fig. 3.

Fig. 3

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Dp116 prolongs the heart periods (HPs). (A–C) Bar graph representations of changes in HPs, systolic and diastolic intervals of young 1- and 5-week-old flies. Note that Dp116 increased the HPs, mainly by prolonging the diastolic interval. Error bars are SEM, N = 20 flies per genotype and age. (D) HP histograms obtained from 30 s optical recordings indicate the distribution of HPs for each fly. Individual data points are plotted, illustrating HP variability for wildtype, Dys−/− and the rescued Dys−/− hearts. The vertical line through the peak of the heart period distributions is to facilitate comparison between the different plots.

3.4. Nitric oxide synthase (Nos) maintains youthful heart function of Dys−/− flies during aging

The findings with the micro-Dys genes ΔH2-R19/ΔCT, ΔR4-23/ΔCT and the Dp116 suggest that the mechanical function of Dys is important to normalize heart diameters and systolic function, whereas the signaling function of Dys, in addition to restoring the diameters and fractional shortening, modulates the heart rate when expressed in Dys−/− mutant background. To further investigate the signaling function of Dys in heart performance, we decided to investigate the role of nitric oxide synthase (Nos) in regulating heart function in wildtype and Dys−/− mutant backgrounds. Nos produces nitric oxide (NO), a signaling molecule known to modulate cardiac function (Balligand et al., 1993). In mammals, the NO role in cardiac function is more complex, since there are three Nos genes with autocrine and paracrine effects (Barouch et al., 2002; Vila-Petroff et al., 1999).

To study the relationship between Dys and Nos signaling function in heart performance and with aging, we have determined the dynamic heart properties of single and combined mutations of Nos and Dys. First, we used RNAi knockdown of transcripts for the single Drosophila Nos gene (NosRNAi) in cardiomyocytes. We found an increase in the HPs at 1 and 5 weeks of age (Fig. 4A), primarily due to prolonged diastolic intervals in young and old flies and to systolic intervals in aged individuals (Fig. 4B, C). Next, we overexpressed wildtype Nos cDNA and found a decrease in HPs, compared with controls (Fig. 4A), mainly due to decreased diastolic intervals (Fig. 4B, C). Taken together, these data suggest that Nos modulates the Drosophila heart rate. To further determine Nos effect in Dys−/− mutants, we made genetic combinations of both genes, i.e. modulating Nos function in Dys deficiency background (Dys−/−; Hand-Gal4 > NosRNAi or Dys−/−; Hand-Gal4 > Nos). We found that either homozygous or heterozygous Dys deficiency reversed the HP increase due to cardiac Nos knockdown in young and old flies, by shortening both diastolic and systolic intervals (Fig. 4A–C). However, the double mutant Dys−/−; Hand-Gal4 > NosRNAi does not survive to 5 weeks of age, indicating that Nos expression is important for long-term survival of Dys−/− flies. Interestingly, the shorter heart periods observed with Nos overexpression tended to be further reduced in a Dys deficiency background (Dys−/−; Hand-Gal4 > Nos), due to decreased diastolic and systolic intervals (Fig. 4A–C). Nos overexpression is beneficial for the Dys−/− mutant, so that the heart performance of old Dys−/− flies was similar to that of young flies. The variability in the heart periodicity is quantified using the HP standard deviation as an arrhythmia index (AI) (Supplemental Fig. S3A). The AI for Dys−/− was significantly reduced at an older age due to overexpression of Nos (Supplemental Fig. S3A). In addition, in old flies Nos knockdown worsened the heart systolic diameters of Dys+/−, and Nos gain of function normalized the fractional shortening of 5 week old Dys−/− (Fig. S3B), implying that gain of Nos function is cardioprotective upon loss-of-Dys function. However, further studies are needed to determine the specific effects of Nos manipulation on cardiac contractility. Taken together, these results demonstrate that Nos is a key regulator of heartbeat, and acts in a cardiomyocyte-autonomous and age-dependent manner.

Fig. 4.

Fig. 4

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Nitric oxide synthase (Nos) regulates the Drosophila heart beat. (A–C) Heart beat parameters in 1- and 5-week-old flies. Cardiac RNAi knockdown of Nos shows significant increase in HPs and diastolic intervals. An effect reversed in Dys−/− mutants. Error bars are SEM. N = 20 flies per genotype and age. Differences were estimated by one-way ANOVA analysis (*p < 0.05; **p < 0.005; ***p < 0.0005). (D) Representative confocal image of adult cardiomyocyte stained with α-actinin (D) and NOS antibody (D′) from hearts overexpressing Nos. Nos protein is found in a doublet of bands on each side of α-actinin (D″). (E) Schematic outline of the full-length Dys, micro-Dys and Dp116 and their interaction with other subcellular components. Dys full-length binds to F-actin and to the DGC complex. The micro-Dys binding to F-actin and not interacting with Nos is functional and rescues the heart cell contractility. Similarly, the Dp116 which does not bind F-actin but links the other DGC proteins is efficient in reinstating the cardiac function. Note that Nos in the sarcolemma is what is known from vertebrate cardiomyocyte biochemistry. Note the new Nos pattern close to the Z-lines. Dg: Dystroglycan, SGs: sarcoglycan complex, Dbr: dystrobrevin, Syn: syntrophin, N: NH2-terminal actin-binding domain, CR: cysteine-rich domain, CT: carboxy-terminal domain.

Using two different anti-Nos antibodies we could detect weak Nos staining in the adult heart cardiomyocytes and strong Nos expression in body wall muscles and neurons innervating cardiac cells (Fig. S3C–E). Also, upon overexpressing Nos we observed a striated sarcomeric pattern organized in a doublet of bands on either side of the Z-lines stained with α-actinin antibodies (Fig. 4D–D″), which may explain increased heart rate in Hand-Gal4 > Nos (Fig. 4A). These observations are evidence that Nos protein is associated with sarcomeres, and might thus regulate the contractility of cardiac muscle, improving heart performance in aged Dys−/− mutant flies.

4. Discussion

Cardiomyopathy is a major health threat to DMD and BMD patients. Focusing on the gene therapy strategy to redress cardiac pump failure is of great importance to alleviate the severity of this heart disease. In the present study, we evaluated truncated Dys genes for their potential to rescue the heart phenotypes in Drosophila Dys−/− mutants. We found that both micro-Dys constructs with a predicted mechanical role and Dp116-Dys with a predicted signaling role can markedly reverse morphological and functional features of dystrophic hearts. Specifically, we highlight the beneficial effect of Dp116 in rescuing the Dys−/− heart abnormalities even in the absence of a predicted actin cytoskeleton link (Fig. 4E).

The expression of micro-Dys in mdx mice results in cardiac histopathology correction and partial normalization of heart function (Bostick et al., 2009, 2011; Townsend et al., 2007), consistent with our data, which also demonstrate partially normalized heart contractility of young and aged dystrophic flies. Both micro-Dys proteins fail to correct the increased heart rate of old Dys−/− flies, again consistent with ΔH2-R19 also having no effect on tachycardia in mdx mice (Bostick et al., 2009). This suggests that micro-Dys functions in the fly heart mirror in many aspects their functional capacities in the mdx mouse. Interestingly, in Drosophila, the micro-Dys rescue of heart function also includes restoration of cardiomyocyte myofibril integrity. Whether a residual myofibrillar mis-orientation is a similar phenomenon to that of micro-Dys ΔR4-23/ΔCT generating ringed fibers in skeletal muscle of mdx mice (Banks et al., 2010) remains to be established.

The expression of Dp116 does not seem to ameliorate the dystrophic phenotypes of mdx mice or the extensive muscle degeneration in mdx; utrn−/− double knockout mice, but it improves their mobility and lifespan (Judge et al., 2006, 2011), suggesting that predicted non-mechanical functions of Dys are indeed important. Also, Dys isoform Dp71 (predicted to have a non-mechanical function) is not sufficient to prevent the mdx;utrn−/− cardiomyopathy, probably because Dp71, but not Dp116, lacks the entire rod domain as well as a functional WW domain, and only weakly associates with the sarcolemma (Greenberg et al., 1994; Hainsey et al., 2003). We showed that the Dp116 ameliorates the Dys−/− flies’ cardiomyopathy and is effective in restoring the cytoarchitectural myofibrils, also suggesting an important signaling role of Dys. It will be interesting to examine the rescue abilities further in the future with other assays, for example the atomic force microscopy-based analysis to measure the passive mechanical stiffness of the cardiomyocytes (Kaushik et al., 2011).

To further explore the non-mechanical role of Dys, we analyzed Nos, known to be involved in Dys-complex signaling and the progression of myopathy. In vertebrate heart muscle, Nos modulates excitation–contraction coupling and thus myocardial function (Ziolo et al., 2008), but its role in regulating heart rhythm remains controversial (Barouch et al., 2002; Khan et al., 2003; Sears et al., 2003). Unexpectedly, genetic disruption of all Nos genes [neuronal (nNos), inducible (iNos) and endothelial (eNos)] results in viable mice with left ventricular hypertrophy and diastolic dysfunction, consistent with previous studies indicating that No affects cardiac remodeling (Janssens et al., 2004; Shibata et al., 2010). We found that cardiac-specific RNAi knockdown of Nos resulted in lower heart rates, due to enlarged relaxation periods. Recent studies revealed that myocardial nNos promotes the [Ca2+]i decay and relaxation by stimulating sarcoplasmic reticulum (SR) Ca2 + reuptake (Tong et al., 2010; Zhang et al., 2008), possibly in close proximity to SR and Z-lines as our data suggest (Fig. 4D). One of the Nos targets could be the sarcoplasmic/endoplasmic reticulum Ca2 ± ATPase (SERCA). In intact arteries, NO induced post-translational modifications such as S-glutathiolation of SERCA, thus increasing SERCA activity and enabling Ca2 ± uptake by the SR (Adachi et al., 2004). Alternatively, Nos may also directly affect transcriptional activation of effector genes (Caceres et al., 2011). Human and mice ventricular myocardial sections show a similar SR-related pattern of nNos (Ramachandran et al., 2013; Xu et al., 1999). Interestingly, the slower heart rate of NosRNAi flies was reversed in Dys−/− mutant background, so that Dys−/−; Hand > NosRNAi flies exhibited a significant decrease in both diastolic and systolic intervals. These results allow the hypothesis that Dys and Nos differentially regulate a common but still unknown target involved in Ca2+ homeostasis and in modulating heart periods.

In conclusion, our findings reveal an age-related cardioprotective effect of truncated Dys proteins, including Dp116 lacking the actin cytoskeleton link, raising intriguing possibilities of Dys signaling functions that warrant further investigation in a less complex cardiac model such as Drosophila. Our study also supports the importance of micro-Dys based gene therapy approaches in improving cardiac performance in DMD patients.

Supplementary Material

Supplemental Data

Acknowledgments

This work was supported by the Muscular Dystrophy Association, the NIA and NHLBI of the National Institutes of Health and the Ellison Medical Foundation (to R.B.), by NIH (to J.S.C.) and by ‘Conseil Régional Auvergne’ (to O.T-L).

Abbreviations

Dys

Dystrophin

DMD

Duchene muscular dystrophy

BMD

Becker muscular dystrophy

XLDCM

X-linked dilated cardiomyopathy

DCM

Dilated cardiomyopathy

AAV

Adeno-associated virus

Dg

Dystroglycan

CR

Cysteine-rich domain

nNos

Neuronal nitric oxide synthase

Grb2

Growth factor receptor-bound protein 2

DGC

Dystrophin-glycoprotein complex

Utrn

Utrophin

Syn

Syntrophin

Dbr

Dystrobrevin

Dg

Dystroglycan

HPs

Heart periods

CT

Carboxy-terminal domain

NO

Nitric oxide

AI

Arrhythmia index

iNos

Inducible Nos

eNos

Endothelial Nos

SR

Sarcoplasmic reticulum

Footnotes

Conflict of interest

The authors declare that they have no conflict of interest.

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