Duchenne muscular dystrophy (DMD), one of the most serious genetic childhood diseases, is characterized by progressive weakness and atrophy after repetitive cycles of degeneration and regeneration due to the lack of dystrophin (Bell and Conen, 1968; Kinali et al., 2011; Vohra et al., 2015). The dystrophin-deficient mdx mouse also suffers from repetitive muscle degeneration (Pastoret and Sebille, 1995), which is one of the reasons why it has been extensively used as a model of human DMD. A key feature of mdx mice is that muscles composed of predominantly fast-twitch fibers are hypersensitive to losing strength in response to eccentric (ECC) contractions (Head et al., 1992; Moens et al., 1993; Lindsay et al., 2019; Kiriaev et al., 2021). Numerous laboratories have used ex vivo, in situ, or in vivo muscle preparations to demonstrate that loss of strength during and immediately following ECC contractions is 20–60% greater in mdx compared with wild-type (WT) mice (Moens et al., 1993; Petrof et al., 1993; Brooks, 1998; Call et al., 2011; Pratt et al., 2013, 2015; Baumann et al., 2020; Lindsay et al., 2020). Susceptibility to ECC contraction-induced strength loss in mdx muscles has therefore become a standard outcome measure in preclinical studies to assess disease severity and the efficacy of potential therapies for DMD. However, despite the widespread use of ECC contraction protocols, the muscular dystrophy field remains uncertain what underlying mechanism initiates the immediate strength loss in mdx muscle. Function of several cellular and subcellular structures in muscle that are necessary for contraction and force generation have been reported to be disrupted following ECC contractions in mdx muscle, including the neuromuscular junction (NMJ) (Pratt et al., 2013, 2015), plasmalemma (Petrof et al., 1993; Call et al., 2013; Roy et al., 2016), ryanodine receptor (Bellinger et al., 2009; Lindsay et al., 2020), sarco/endoplasmic reticulum Ca²⁺-ATPase (Mázala et al., 2015; Lindsay et al., 2020), and myofibrillar proteins (Blaauw et al., 2010).

All the aforementioned structures are putative sites contributing to contraction-induced strength loss in mdx muscle (see review by Warren et al. (2001) for WT muscle). However, we posit that strength loss in mdx muscle is initially triggered, in large, by plasmalemmal inexcitability (Call et al., 2013; Baumann et al., 2020). Three main arguments can be made to support this proposition. First, it has repeatedly been demonstrated that in vivo ECC contraction-induced reductions in peak torque in mdx muscle parallel reductions of M-wave root-mean-square (M-wave RMS; i.e., a measure of plasmalemmal excitability; Call et al., 2013; Roy et al., 2016; Baumann et al., 2020). Second, loss of M-wave RMS corresponds with the resting membrane potential of fibers becoming more positive (i.e., depolarized; Call et al., 2013). Finally, M-wave RMS and the resting membrane potential in WT muscle fibers following ECC contractions are unchanged or only marginally decreased (Warren et al., 1999; Call et al., 2013; Baumann et al., 2020). These three points suggest ECC contractions depolarize mdx fibers leaving them inexcitable (i.e., unable to generate and/or conduct action potentials). Fewer action potentials will be observed as reductions in M-wave RMS and, inevitability, the muscle’s force generating capacity (Call et al., 2013; Baumann et al., 2020). Surprisingly, despite peak isometric torque and M-wave RMS being reduced approximately 40–60% after a single bout of 50–100 maximal ECC contractions, mdx muscle exhibits a remarkable ability to recover, as both variables return to baseline within 2weeks (Call et al., 2013; Baumann et al., 2020). These results indicate dystrophin may be necessary for excitation to occur at the plasmalemma during a series of ECC contractions, but not essential for complete recovery of plasmalemma electrophysiological function or isometric strength (Call et al., 2013; Baumann et al., 2020).

Dystrophin is one of the many proteins that make up the dystrophin glycoprotein complex (DGC). The DGC is also composed of cytoplasmic, transmembrane, and extracellular proteins including the sarcoglycans, dystroglycans, dystrobrevins, syntrophins, sarcospan, caveolin-3, and neuronal nitric oxide (NO) synthase (Straub and Campbell, 1997; Lapidos et al., 2004). It has become increasingly clear that the DGC holds both structural and signal transduction properties (Lapidos et al., 2004; Constantin, 2014; Garbincius and Michele, 2015) and provides a strong mechanical link from the intracellular cytoskeleton to the extracellular matrix (Rybakova et al., 2000). In our most recent publication, we suggested that future work will need to determine how DGC proteins, in addition to dystrophin, contribute to electrophysiological (dys)function of the plasmalemma (Baumann et al., 2020).

To advance on our suggested work, here, we strategically selected four different mouse lines that possess varying contents of dystrophin and DGC proteins as well as functionality. WT mice served as healthy controls while mdx, mL172H and Sgcb^−/− mice represented lines mimicking Duchenne, Becker and Limb-girdle Type 2E muscular dystrophies, respectively (Ng et al., 2012; McCourt et al., 2018). As mentioned, mdx mice are not only dystrophin deficient but also have reduced contents of other DGC proteins (Ohlendieck and Campbell, 1991). The transgenic mL172H mouse expresses dystrophin with an L172H missense mutation associated with Becker muscular dystrophy on the mdx background (McCourt et al., 2018). The mutant L172H dystrophin is expressed at ~40% of WT and localizes to the plasmalemma but only partially restores the expression of other DGC components (McCourt et al., 2018). In contrast, the Sgcb^−/− mouse expresses WT levels of dystrophin at the plasmalemma and retains the DGC but lacks the entire sarcoglycan complex due to the absence of β-sarcoglycan (Araishi et al., 1999; Durbeej et al., 2000). Using these mouse lines, which retain varying degrees of dystrophin and DGC protein contents and functionality, we hypothesized that in order to attenuate ECC contraction-induced plasmalemmal inexcitability, dystrophin would not only need to be present but also “functional” (Baumann et al., 2020). Stated differently, maintaining electrophysiological function of the plasmalemma would not solely be dependent on having dystrophin but rather having dystrophin working collectively with other DGC proteins. Therefore, the purpose was to determine if maintenance of plasmalemmal excitability was dependent on completely intact and fully functional DGCs. To accomplish this, we measured M-wave RMS and torque before, during and after a single bout of ECC contractions in WT, mdx, mL172H, and Sgcb^−/− mice.

The purpose of this study was to determine if loss of plasmalemmal excitability is dependent on having completely intact and fully functional DGCs rather than just dystrophin content. By comparing mouse lines that had varying amounts and functionality of dystrophin and other DGC proteins, we observed three primary findings. First, M-wave RMS does not change during or after a bout of ECC contractions in WT mice, meaning that when all DGCs are intact, plasmalemmal action potential generation and conduction are maintained. Second, Sgcb^−/− mice lose M-wave RMS due to ECC contractions, which parallels that observed in mdx mice, indicating that deficiency of the DGC protein β-sarcoglycan is as disruptive to plasmalemmal excitability as is dystrophin deficiency. Third, mL172H mice are also susceptible to ECC-induced reductions in M-wave RMS yet show marginal protection when compared to mdx or Sgcb^−/− mice, suggesting some functionally intact DGCs are better than none. These results support our hypothesis that maintaining electrophysiological function of the plasmalemma during ECC contractions is dependent on dystrophin working collectively with other DGC proteins.

Mdx, mL172H, and Sgcb^−/− mouse lines model Duchenne, Becker, and Limb-girdle Type 2E muscular dystrophies, respectively. Importantly, we specifically selected these three dystrophic mouse lines to assess how altered expression level and functionality of dystrophin and the DGC impacts plasmalemmal excitability during and after ECC contractions. Mdx mice are dystrophin deficient and have reduced contents of other DGC proteins (Ohlendieck and Campbell, 1991). For instance, we found that β-sarcoglycan was 58% lower in mdx muscle compared to that of WT muscle. Sgcb^−/− mice are β-sarcoglycan deficient and lack the entire sarcoglycan complex, but express WT levels of plasmalemmal dystrophin and DGCs (Araishi et al., 1999; Durbeej et al., 2000). The mL172H mouse expresses a mutated form of dystrophin that is approximately 30–40% of WT (present study; McCourt et al., 2018). However, expression of L172H dystrophin does not completely restore other DGC components to WT levels yet properly localizes DGC components to the plasmalemma (McCourt et al., 2018). Regardless of how dystrophin or the DGC was perturbed, all dystrophic lines (mdx, mL172H, and Sgcb^−/−) were hypersensitive to ECC contraction-induced strength loss when compared to WT mice. We posit that these strength deficits were due to reductions in plasmalemmal excitability that stemmed from only partially complete DGCs or complete lack thereof.

Various cytoplasmic, transmembrane, and extracellular proteins make up the DGC including dystrophin, sarcoglycans, dystroglycans, dystrobrevins, syntrophins, sarcospan, caveolin-3, and neuronal nitric oxide (NO) synthase (Straub and Campbell, 1997; Lapidos et al., 2004). Functionally, the DGC provides a strong mechanical link from the intracellular cytoskeleton to the extracellular matrix (Rybakova et al., 2000) and holds both structural and signal transduction properties (Lapidos et al., 2004; Constantin, 2014; Garbincius and Michele, 2015) making it hard to discern how the DGC specifically influences plasmalemmal excitability. Loss of M-wave RMS could be due to transmission failure at the NMJ or an impaired ability to generate and/or conduct an action potential along the plasmalemma. NMJ transmission failure is largely based on histological evidence demonstrating that NMJ morphology is altered after performing ECC contractions in mdx mice but maintained in WT mice (Pratt et al., 2013, 2015). Conversely, loss of M-wave RMS due to plasmalemmal dysfunction is based on data obtained from mdx muscle fibers, in which approximately 50% were more positive than −55mV compared with only 7% from that of WT muscle fibers immediately after a bout of ECC contractions (Call et al., 2013). Skeletal muscle fibers are thought to be rendered inexcitable and contribute minimally to force development when depolarized above −55mV (Renaud and Light, 1992; Cairns et al., 1995, 1997). Although it is currently unknown how ECC contractions depolarize mdx muscle fibers (and likely mL172H and Sgcb^−/− muscle fibers), putative mechanisms are loss of physical integrity of the plasmalemma as indicated by Evans Blue Dye (Lindsay et al., 2020), the development of branched fibers in dystrophic muscle (Chan et al., 2007; Head, 2010; Kiriaev et al., 2021), and/or dysfunction of ion channels (Yeung et al., 2005; Whitehead et al., 2006). For instance, it has been proposed that ECC contractions disrupt plasmalemmal ion channel function in branched fibers that results in ion flux, subsequently increasing intracellular Na⁺ and Ca²⁺ concentrations (Chan et al., 2007; Head, 2010). Future investigations will need to determine if the DGC (or lack thereof) results in branched fibers and if so, mechanistically how branched fibers contribute to NMJ transmission failure or the inability to generate and/or conduct an action potential along the plasmalemma.

While out of the scope of the present study, disruption downstream of the plasmalemma may also occur in dystrophic muscle due to ECC contractions. There is ex vivo evidence suggesting that function of the sarcoplasmic reticulum (SR) and myofibrillar apparatus are both involved in ECC-induced strength loss in mdx muscle (Bellinger et al., 2009; Blaauw et al., 2010; Morine et al., 2010; Lindsay et al., 2020). A caveat to ex vivo approaches is that plasmalemmal action potential generation and conduction is typically bypassed using these preps. Thus, SR dysfunction, loss of myofibrillar Ca²⁺ sensitivity, or structural damage to contractile proteins detected ex vivo in injured mdx muscle would not be detectable through in vivo measurements because loss of plasmalemmal excitability precedes them in the excitation-contraction coupling process.

In closing, we previously theorized that in order to attenuate ECC contraction-induced plasmalemmal inexcitability in mdx muscle, dystrophin would not only need to be present but also assembled into an intact and functional DGC (Baumann et al., 2020). Here, we confirm that even with WT levels of dystrophin in skeletal muscle, loss of plasmalemmal electrophysiological function will still occur during maximal ECC contractions if the DGC is otherwise disrupted (i.e., as in the Sgcb^−/− mouse). Moreover, although a certain degree of protection is apparent in muscle with partially functional DGCs (i.e., the mL172H mouse), it remains susceptible to ECC contraction-induced plasmalemmal inexcitability. Our results highlight the significant role the DGC plays in maintaining plasmalemmal electrophysiological function and that a collective synergism (via each DGC protein) is required for this complex to function properly.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by the Institutional Animal Care and Use Committees at the University of Minnesota.

CB, AL, GW, and DL conceived and designed the analysis. CB and SS collected the data and performed the analysis. CB, SS, JE, and DL contributed data or analysis tools. CB, AL, and SS wrote the paper. CB, AL, SS, JE, GW, and DL revised and approved the paper. All authors contributed to the article and approved the submitted version.

This work was funded by the Research Endowment from the American College of Sports Medicine Foundation (to CB), a grant from the University of Minnesota Bob Allison Ataxia Research Center (to DL), and grants from the National Institutes of Health (T32-AG029796 and T32-AR007612 to CB and R01-AR042423 to JE).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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