nNOS plays a pivotal regulatory role in skeletal muscle physiology and disease (1
). Interestingly, nNOS function depends on its location at the sarcolemma. Loss of sarcolemmal nNOS has been shown to induce functional ischemia and exaggerate exercise-associated fatigue (2
). For these reasons, there has been a great interest in understanding the mechanisms involved in anchoring nNOS to the sarcolemma.
Studies in the mid-1990s suggested that the correct positioning of nNOS requires the DGC, a multimeric protein complex consisting of dystrophin, dystroglycans, sarcoglycans, sarcospan, syntrophin, and dystrobrevin. Subsequent biochemical, structural, and genetic analyses demonstrated an essential role for syntrophin (7
). In particular, the PDZ domain of syntrophin forms a complex with the PDZ domain of nNOS (17
). At the same time, it was suggested that nNOS recruitment might require dystrophin. Since the dystrophin C-terminal domain contains a syntrophin binding site, the involvement of dystrophin seems quite logical. Surprisingly, sarcolemmal nNOS expression is not altered in mdx
mice that express C-terminal domain–truncated dystrophins (15
). Furthermore, even in the presence of an intact C-terminal domain, nNOS is not detected at the sarcolemma (21
). Taken together, these findings indicate that the dystrophin C-terminal domain may, at most, play a nominal role in nNOS recruitment.
A more puzzling issue is that nNOS is not always present on the sarcolemma even when syntrophin is there (16
). Apparently, additional help is needed to anchor nNOS to the sarcolemma. Several candidates have been proposed. These include other proteins in the DGC or other proteins that interact with the DGC, such as aquaporin-4, sodium channel, calcium pump, and caveolin-3 (32
). Unfortunately, none of these have been confirmed.
Different regions in the dystrophin N-terminal and rod domains (such as exons 3–7 or 8, 10–42, 45, 45–47, and 51–52) have also been suggested to help recruit nNOS to the sarcolemma (21
). Nevertheless, defining the nNOS recruiting domain in dystrophin has proven to be a quite challenging task. Several lines of evidence suggest that dystrophin may not bind to nNOS directly. First, it has been shown that nNOS interacts with other proteins mainly through the PDZ–PDZ domain interaction (36
). However, dystrophin does not contain a classic PDZ domain. Second, 2 independent studies have failed to detect a direct interaction between full-length dystrophin and nNOS (8
). Proving an indirect interaction is technically much more difficult. Third, it is not clear whether the dystrophin-nNOS interaction (assuming there is one) depends on a single defined region or several discontinuous regions in dystrophin.
In this study, we took a systemic approach to map the nNOS localization domain in dystrophin. We started with the DH2–R19 minigene (26
). We have previously confirmed that this minigene cannot restore sarcolemmal nNOS (23
). Based on this information, we hypothesized that the H2-to-R19 region should contain the domain(s) required for nNOS recruiting. By in vivo plasmid transfection and AAV-mediated gene transfer, we narrowed down the nNOS recruiting domain to R16/17 (Figures and , Supplemental Figures 2–4, and Table ). We found that these 2 repeats were necessary to anchor nNOS to the sarcolemma. Furthermore, R16/17-mediated nNOS recruitment was not dependent on the flanking repeats or the dystrophin C-terminal domain. Interestingly, we also observed an interaction between the nNOS PDZ domain and R16/17, although its strength was lower than that of the interaction between the nNOS PDZ domain and the syntrophin PDZ domain (Figure , D and E). Based on these results, we here propose a novel nNOS recruitment model (Figure ). According to this model, nNOS localization requires 2 independent interactions, one between R16/17 and the nNOS PDZ domain and the other between the syntrophin PDZ domain and the nNOS PDZ domain.
Schematic outline of sarcolemmal nNOS recruitment by an R16/17-containing microdystrophin gene.
This model raises several interesting questions. First, how can a single nNOS PDZ domain interact with 2 partners at the same time? A ternary complex involving the nNOS PDZ domain has been demonstrated before (34
). However, in this type of the complex, the nNOS PDZ domain only interacts with one partner (such as CAPON and postsynaptic density proteins). This intermediate partner then recruits the third partner to the complex (such as small G protein Dexras 1, synapsins, and NMDA receptor) (34
). The classical PDZ domain consists of 2 α helices (αA and αB) and 6 β-sheets (from βA to βF). αB and βB form a groove for ligand binding (37
). The nNOS PDZ domain includes the first 150 N-terminal residues of nNOS. Amino acids 1–100 express the classic groove structure for ligand binding. The next 30 residues form a unique β-finger (17
). Interestingly, the syntrophin PDZ domain only binds to the β-finger. This leaves the nNOS PDZ groove unoccupied (17
). Thus, there is a structural basis for the nNOS PDZ domain to bind to both syntrophin and R16/17 simultaneously (38
Second, R16/17 does not seem to be a good ligand for the nNOS PDZ groove. The short C-terminal peptide motif and the internal peptide motif are the classical ligands for the PDZ groove (37
). Dystrophin does not carry these motifs. It is currently unclear how spectrin-like repeats may interact with the PDZ domain. Interestingly, such interaction has also been observed in another PDZ domain–containing protein. Xia et al. have demonstrated that the PDZ domain of the actinin-associated LIM protein binds to spectrin-like repeats in α-actinin-2 (39
). Interestingly, this interaction also requires 2 complete repeats (39
). Taking together, these findings reveal a new recognition mode for the PDZ domain. The PDZ domain is one of the most frequently used scaffolding modules. Approximately 400 PDZ domains have been identified in the human genome. The identification/confirmation of spectrin-like repeats as a PDZ domain ligand sheds new light on our understanding of protein-protein interaction and oligomerization.
R16/17 is encoded by exons 41–46 (40
). This explains the findings seen in the majority of the clinical cases. In these patients, the loss of sarcolemmal nNOS correlates with mutations between exons 41 and 46 (21
). However, sarcolemmal nNOS is also missing in some patients who were reported to carry mutations in other regions of the dystrophin gene, such as exons 3–8 and 48–52 (21
). There are at least 2 possibilities. Spectrin-like repeats are modular repeating elements. Each repeat is composed of 3 α-helices linked by turns (41
). However, the structural unit of the dystrophin rod domain is not based on individual repeats. Instead, it is believed that the biophysical unit is formed by adjacent repeats in a nested manner (Figure B) (42
). Disruption of one repeat may alter structural conformation of the nearby units and influence the correct phasing of the entire rod domain (43
). Hence, mutations in other exons may alter the noncanonical PDZ groove binding motif formed by R16/17. Alternatively, it is also possible that these patients carry additional small mutations (such as single nucleotide change) between exons 41 and 46.
The ultimate goal of our study is to develop an effective gene therapy to treat all affected muscles in DMD/BMD. AAV is the only viral vector proven to be capable of body-wide gene delivery (27
). However, AAV cannot carry the full-length dystrophin coding sequence. Minimizing the dystrophin gene offers a valid avenue to circumvent this problem (26
). A variety of different minigenes and microgenes have been developed. Unfortunately, none of the abbreviated genes have been conclusively shown to recruit nNOS to the sarcolemma.
Based on the finding that R16/17 is sufficient to restore sarcolemmal nNOS, we generated a new microdystrophin gene, DR2–15/DR18–23/DC. This microgene is similar to our previously reported DR4–23/DC microgene, except that R2–H2 is replaced by R16/17 (Figure A) (16
). Local and systemic gene transfer studies were performed in newborn and adult mouse DMD models. In mdx
, and MyoD/dystrophin double-knockout mice, the R16/17-containing microgene not only restored sarcolemmal nNOS, but also effectively reduced muscle histopathology and enhanced sarcolemma integrity (Figure , Figure , and Supplemental Figures 4 and 5). Furthermore, muscle strength and resistance to contraction-induced injury were improved to levels comparable to those resulting from DR4–23/DC microgene treatment (Figure B and Supplemental Figure 6).
One caveat with the microgenes is the uncertainty of their function in the canine animal model and human patients (45
). On the other hand, the minigenes are derived from a remarkably functional template isolated from a patient with very mild BMD (46
). They also restore muscle-specific forces to normal levels (Figure D) (26
). Importantly, the minigenes can be delivered by trans
-splicing AAV vectors to all muscles in the body (25
). From this standpoint, minigene therapy could be more effective than microgene therapy. To determine the therapeutic advantage of the R16/17-containing minigene, we next compared muscle force, femoral artery hemodynamics, muscle perfusion, and treadmill running in DH2–R15 and DH2–R19 transgenic mice (Figures – and Supplemental Figures 7, 8, and 10). Both transgenic strains were able to recover wild-type-level muscle force (Figure D). However, only the R16/17-containing minigene transgenic mice (DH2–R15) showed significantly improved perfusion in contracting muscle (Figure , Figure , and Supplemental Figure 8). Furthermore, restoring sarcolemmal nNOS by transfection of the DH2–R15 minigene significantly enhanced exercise performance and prevented ischemic damage (Figure and Supplemental Figure 10).
Taken together, our studies provide evidence that R16/17 is essential for anchoring nNOS to the sarcolemma. The R16/17-containing mini-/microgenes may be more effective for DMD gene therapy than all previously described mini-/microgenes. Our findings also shed new light on the molecular mechanisms of nNOS PDZ domain–mediated scaffolding. Future studies on how R16/17 coordinates with syntrophin to bring nNOS to the sarcolemma may unravel yet-unrecognized aspects of this fascinating biological process.