The slow-release adiponectin analog ALY688-SR modifies early-stage disease development in the D2.mdx mouse model of Duchenne muscular dystrophy.

Fibrosis is associated with respiratory and limb muscle atrophy in Duchenne muscular dystrophy (DMD). Current standard of care partially delays the progression of this myopathy but there remains an unmet need to develop additional therapies. Adiponectin receptor agonism has emerged as a possible therapeutic target to lower inflammation and improve metabolism in mdx mouse models of DMD but the degree to which fibrosis and atrophy are prevented remain unknown. Here, we demonstrate that the recently developed slow-release peptidomimetic adiponectin analog, ALY688-SR, remodels the diaphragm of murine model of DMD on DBA background (D2.mdx) mice treated from days 7-28 of age during early stages of disease. ALY688-SR also lowered interleukin-6 (IL-6) mRNA but increased IL-6 and transforming growth factor-ß1 (TGF-ß1) protein contents in diaphragm, suggesting dynamic inflammatory remodeling. ALY688-SR alleviated mitochondrial redox stress by decreasing complex I-stimulated H2O2 emission. Treatment also attenuated fibrosis, fiber type-specific atrophy, and in vitro diaphragm force production in diaphragm suggesting a complex relationship between adiponectin receptor activity, muscle remodeling, and force-generating properties during the very early stages of disease progression in murine model of DMD on DBA background (D2.mdx) mice. In tibialis anterior, the modest fibrosis at this young age was not altered by treatment, and atrophy was not apparent at this young age. These results demonstrate that short-term treatment of ALY688-SR in young D2.mdx mice partially prevents fibrosis and fiber type-specific atrophy and lowers force production in the more disease-apparent diaphragm in relation to lower mitochondrial redox stress and heterogeneous responses in certain inflammatory markers. These diverse muscle responses to adiponectin receptor agonism in early stages of DMD serve as a foundation for further mechanistic investigations.NEW & NOTEWORTHY There are limited therapies for the treatment of Duchenne muscular dystrophy. As fibrosis involves an accumulation of collagen that replaces muscle fibers, antifibrotics may help preserve muscle function. We report that the novel adiponectin receptor agonist ALY688-SR prevents fibrosis in the diaphragm of D2.mdx mice with short-term treatment early in disease progression. These responses were related to altered inflammation and mitochondrial functions and serve as a foundation for the development of this class of therapy.

Measuring Diaphragm Thickness and Function Using Point-of-Care Ultrasound.

The diaphragm is the main component of the respiratory muscle pump. Diaphragm dysfunction can cause dyspnea and exercise intolerance, and predisposes affected individuals to respiratory failure. In mechanically ventilated patients, the diaphragm is susceptible to atrophy and dysfunction through disuse and other mechanisms. This contributes to failure to wean and poor long-term clinical outcomes. Point-of-care ultrasound provides a valid and reproducible method for evaluating diaphragm thickness and contractile activity (thickening fraction during inspiration) that can be readily employed by clinicians and researchers alike. This article presents best practices for measuring diaphragm thickness and quantifying diaphragm thickening during tidal breathing or maximal inspiration. Once mastered, this technique can be used to diagnose and prognosticate diaphragm dysfunction, and guide and monitor response to treatment over time in both healthy individuals and acute or chronically ill patients.

Memory impairment in the D2.mdx mouse model of Duchenne muscular dystrophy is prevented by the adiponectin receptor agonist ALY688.

NEW FINDINGS: What is the central question of this study? Can adiponectin receptor agonism improve recognition memory in a mouse model of Duchenne muscular dystrophy? What is the main finding and its importance? Short-term treatment with the new adiponectin receptor agonist ALY688 improves recognition memory in D2.mdx mice. This finding suggests that further investigation into adiponectin receptor agonism is warranted, given that there remains an unmet need for clinical approaches to treat this cognitive dysfunction in people with Duchenne muscular dystrophy. ABSTRACT: Memory impairments have been well documented in people with Duchenne muscular dystrophy (DMD). However, the underlying mechanisms are poorly understood, and there is an unmet need to develop new therapies to treat this condition. Using a novel object recognition test, we show that recognition memory impairments in D2.mdx mice are completely prevented by daily treatment with the new adiponectin receptor agonist ALY688 from day 7 to 28 of age. In comparison to age-matched wild-type mice, untreated D2.mdx mice demonstrated lower hippocampal mitochondrial respiration (carbohydrate substrate), greater serum interleukin-6 cytokine content and greater hippocampal total tau and Raptor protein contents. Each of these measures was partly or fully preserved after treatment with ALY688. Collectively, these results indicate that adiponectin receptor agonism improves recognition memory in young D2.mdx mice.

Mitochondrial creatine sensitivity is lost in the D2.mdx model of Duchenne muscular dystrophy and rescued by the mitochondrial-enhancing compound Olesoxime.

Duchenne muscular dystrophy (DMD) is associated with distinct mitochondrial stress responses. Here, we aimed to determine whether the prospective mitochondrial-enhancing compound Olesoxime, prevents early-stage mitochondrial stress in limb and respiratory muscle from D2.mdx mice using a proof-of-concept short-term regimen spanning 10-28 days of age. As mitochondrial-cytoplasmic energy transfer occurs via ATP- or phosphocreatine-dependent phosphate shuttling, we assessed bioenergetics with or without creatine in vitro. We observed that disruptions in Complex I-supported respiration and mH2O2 emission in D2.mdx quadriceps and diaphragm were amplified by creatine demonstrating mitochondrial creatine insensitivity manifests ubiquitously and early in this model. Olesoxime selectively rescued or maintained creatine sensitivity in both muscles, independent of the abundance of respiration-related mitochondrial proteins or mitochondrial creatine kinase cysteine oxidation in quadriceps. Mitochondrial calcium retention capacity and glutathione were altered in a muscle-specific manner in D2.mdx but were generally unchanged by Olesoxime. Treatment reduced serum creatine kinase (muscle damage) and preserved cage hang-time, microCT-based volumes of lean compartments including whole body, hindlimb and bone, recovery of diaphragm force after fatigue, and cross-sectional area of diaphragm type IIX fiber, but reduced type I fibers in quadriceps. Grip strength, voluntary wheel-running and fibrosis were unaltered by Olesoxime. In summary, locomotor and respiratory muscle mitochondrial creatine sensitivities are lost during early stages in D2.mdx mice but are preserved by short-term treatment with Olesoxime in association with specific indices of muscle quality suggesting early myopathy in this model is at least partially attributed to mitochondrial stress.

Muscle weakness precedes atrophy during cancer cachexia and is linked to muscle-specific mitochondrial stress.

Muscle weakness and wasting are defining features of cancer-induced cachexia. Mitochondrial stress occurs before atrophy in certain muscles, but the possibility of heterogeneous responses between muscles and across time remains unclear. Using mice inoculated with Colon-26 cancer, we demonstrate that specific force production was reduced in quadriceps and diaphragm at 2 weeks in the absence of atrophy. At this time, pyruvate-supported mitochondrial respiration was lower in quadriceps while mitochondrial H2O2 emission was elevated in diaphragm. By 4 weeks, atrophy occurred in both muscles, but specific force production increased to control levels in quadriceps such that reductions in absolute force were due entirely to atrophy. Specific force production remained reduced in diaphragm. Mitochondrial respiration increased and H2O2 emission was unchanged in both muscles versus control while mitochondrial creatine sensitivity was reduced in quadriceps. These findings indicate muscle weakness precedes atrophy and is linked to heterogeneous mitochondrial alterations that could involve adaptive responses to metabolic stress. Eventual muscle-specific restorations in specific force and bioenergetics highlight how the effects of cancer on one muscle do not predict the response in another muscle. Exploring heterogeneous responses of muscle to cancer may reveal new mechanisms underlying distinct sensitivities, or resistance, to cancer cachexia.

Mitochondrial stress responses in Duchenne muscular dystrophy: metabolic dysfunction or adaptive reprogramming?

Mitochondrial stress may be a secondary contributor to muscle weakness in inherited muscular dystrophies. Duchenne muscular dystrophy has received the majority of attention, whereby most discoveries suggest mitochondrial ATP synthesis may be reduced. However, not all studies support this finding. Furthermore, some studies have reported increased mitochondrial reactive oxygen species and propensity for permeability transition pore formation as an inducer of apoptosis, although divergent findings have also been described. A closer examination of the literature suggests the degree and direction of mitochondrial stress responses may depend on the progression of the disease, the muscle type examined, the mouse model used with regard to preclinical research, the precise metabolic pathways in consideration, and in some cases, the in vitro technique used to assess a given mitochondrial bioenergetic function. One intent of this review is to provide careful considerations for future experimental designs to resolve the heterogeneous nature of mitochondrial stress during the progression of Duchenne muscular dystrophy. Such considerations have implications for other muscular dystrophies as well which are addressed briefly herein. A renewed perspective of the term "mitochondrial dysfunction" is presented whereby stress responses might be re-explored in future investigations as direct contributors to myopathy versus an adaptive "reprogramming" intended to maintain homeostasis in the face of disease stressors themselves. In so doing, the prospective development of mitochondrial enhancement therapies can be driven by advances in perspectives as much as experimental approaches when resolving the precise relationships between mitochondrial remodeling and muscle weakness in Duchenne and, indeed, other muscular dystrophies.

Loss of dystrophin expression in skeletal muscle is associated with senescence of macrophages and endothelial cells.

Cellular senescence is the irreversible arrest of normally dividing cells and is driven by cell cycle inhibitory proteins such as p16, p21, and p53. When cells enter senescence, they secrete a host of proinflammatory factors known as the senescence-associated secretory phenotype, which has deleterious effects on surrounding cells and tissues. Little is known of the role of senescence in Duchenne muscular dystrophy (DMD), the fatal X-linked neuromuscular disorder typified by chronic inflammation, extracellular matrix remodeling, and a progressive loss in muscle mass and function. Here, we demonstrate using C57-mdx (8-wk-old) and D2-mdx (4-wk-old and 8-wk-old) mice, two mouse models of DMD, that cells displaying canonical markers of senescence are found within the skeletal muscle. Eight-week-old D2-mdx mice, which display severe muscle pathology, had greater numbers of senescent cells associated with areas of inflammation, which were mostly Cdkn1a-positive macrophages, whereas in C57-mdx muscle, senescent populations were endothelial cells and macrophages localized to newly regenerated myofibers. Interestingly, this pattern was similar to cardiotoxin (CTX)-injured wild-type (WT) muscle, which experienced a transient senescent response. Dystrophic muscle demonstrated significant upregulations in senescence pathway genes [Cdkn1a (p21), Cdkn2a (p16INK4A), and Trp53 (p53)], which correlated with the quantity of senescence-associated ß-galactosidase (SA-ß-Gal)-positive cells. These results highlight an underexplored role for cellular senescence in murine dystrophic muscle.

Mitochondrial bioenergetic dysfunction in the D2.mdx model of Duchenne muscular dystrophy is associated with microtubule disorganization in skeletal muscle.

In Duchenne muscular dystrophy, a lack of dystrophin leads to extensive muscle weakness and atrophy that is linked to cellular metabolic dysfunction and oxidative stress. This dystrophinopathy results in a loss of tethering between microtubules and the sarcolemma. Microtubules are also believed to regulate mitochondrial bioenergetics potentially by binding the outer mitochondrial membrane voltage dependent anion channel (VDAC) and influencing permeability to ADP/ATP cycling. The objective of this investigation was to determine if a lack of dystrophin causes microtubule disorganization concurrent with mitochondrial dysfunction in skeletal muscle, and whether this relationship is linked to altered binding of tubulin to VDAC. In extensor digitorum longus (EDL) muscle from 4-week old D2.mdx mice, microtubule disorganization was observed when probing for α-tubulin. This cytoskeletal disorder was associated with a reduced ability of ADP to stimulate respiration and attenuate H2O2 emission relative to wildtype controls. However, this was not associated with altered α-tubulin-VDAC2 interactions. These findings reveal that microtubule disorganization in dystrophin-deficient EDL is associated with impaired ADP control of mitochondrial bioenergetics, and suggests that mechanisms alternative to α-tubulin's regulation of VDAC2 should be examined to understand how cytoskeletal disruption in the absence of dystrophin may cause metabolic dysfunctions in skeletal muscle.

Sexual dimorphism in human skeletal muscle mitochondrial bioenergetics in response to type 1 diabetes.

Sexual dimorphism in mitochondrial respiratory function has been reported in young women and men without diabetes, which may have important implications for exercise. The purpose of this study was to determine if sexual dimorphism exists in skeletal muscle mitochondrial bioenergetics in people with type 1 diabetes (T1D). A resting muscle microbiopsy was obtained from women and men with T1D (n = 10/8, respectively) and without T1D (control; n = 8/7, respectively). High-resolution respirometry and spectrofluorometry were used to measure mitochondrial respiratory function, hydrogen peroxide (mH2O2) emission and calcium retention capacity (mCRC) in permeabilized myofiber bundles. The impact of T1D on mitochondrial bioenergetics between sexes was interrogated by comparing the change between women and men with T1D relative to the average values of their respective sex-matched controls (i.e., delta). These aforementioned analyses revealed that men with T1D have increased skeletal muscle mitochondrial complex I sensitivity but reduced complex II sensitivity and capacity in comparison to women with T1D. mH2O2 emission was lower in women compared with men with T1D at the level of complex I (succinate driven), whereas mCRC and mitochondrial protein content remained similar between sexes. In conclusion, women and men with T1D exhibit differential responses in skeletal muscle mitochondrial bioenergetics. Although larger cohort studies are certainly required, these early findings nonetheless highlight the importance of considering sex as a variable in the care and treatment of people with T1D (e.g., benefits of different exercise prescriptions).

Neurogranin is expressed in mammalian skeletal muscle and inhibits calcineurin signaling and myoblast fusion.

Calcineurin is a Ca2+/calmodulin (CaM)-dependent phosphatase that plays a critical role in promoting the slow fiber phenotype and myoblast fusion in skeletal muscle, thereby making calcineurin an attractive cellular target for enhancing fatigue resistance, muscle metabolism, and muscle repair. Neurogranin (Ng) is a CaM-binding protein thought to be expressed solely in brain and neurons, where it inhibits calcineurin signaling by sequestering CaM, thus lowering its cellular availability. Here, we demonstrate for the first time the expression of Ng protein and mRNA in mammalian skeletal muscle. Both protein and mRNA levels are greater in slow-oxidative compared with fast-glycolytic muscles. Coimmunoprecipitation of CaM with Ng in homogenates of C2C12 myotubes, mouse soleus, and human vastus lateralis suggests that these proteins physically interact. To determine whether Ng inhibits calcineurin signaling in muscle, we used Ng siRNA with C2C12 myotubes to reduce Ng protein levels by 60%. As a result of reduced Ng expression, C2C12 myotubes had enhanced CaM-calcineurin binding and calcineurin signaling as indicated by reduced phosphorylation of nuclear factor of activated T cells and increased utrophin mRNA. In addition, calcineurin signaling affects the expression of myogenin and stabilin-2, which are involved in myogenic differentiation and myoblast fusion, respectively. Here, we found that both myogenin and stabilin-2 were significantly elevated by Ng siRNA in C2C12 cells, concomitantly with an increased fusion index. Taken together, these results demonstrate the expression of Ng in mammalian skeletal muscle where it appears to be a novel regulator of calcineurin signaling.