The Adiponectin Receptor Agonist, ALY688: A Promising Therapeutic for Fibrosis in the Dystrophic Muscle.

Duchenne muscular dystrophy (DMD) is one of the most devastating myopathies, where severe inflammation exacerbates disease progression. Previously, we demonstrated that adiponectin (ApN), a hormone with powerful pleiotropic effects, can efficiently improve the dystrophic phenotype. However, its practical therapeutic application is limited. In this study, we investigated ALY688, a small peptide ApN receptor agonist, as a potential novel treatment for DMD. Four-week-old mdx mice were subcutaneously treated for two months with ALY688 and then compared to untreated mdx and wild-type mice. In vivo and ex vivo tests were performed to assess muscle function and pathophysiology. Additionally, in vitro tests were conducted on human DMD myotubes. Our results showed that ALY688 significantly improved the physical performance of mice and exerted potent anti-inflammatory, anti-oxidative and anti-fibrotic actions on the dystrophic muscle. Additionally, ALY688 hampered myonecrosis, partly mediated by necroptosis, and enhanced the myogenic program. Some of these effects were also recapitulated in human DMD myotubes. ALY688's protective and beneficial properties were mainly mediated by the AMPK-PGC-1α axis, which led to suppression of NF-κß and TGF-ß. Our results demonstrate that an ApN mimic may be a promising and effective therapeutic prospect for a better management of DMD.

AdipoRon enhances healthspan in middle-aged obese mice: striking alleviation of myosteatosis and muscle degenerative markers.

BACKGROUND: Obesity among older adults has increased tremendously. Obesity accelerates ageing and predisposes to age-related conditions and diseases, such as loss of endurance capacity, insulin resistance and features of the metabolic syndrome. Namely, ectopic lipids play a key role in the development of nonalcoholic fatty liver disease (NAFLD) and myosteatosis, two severe burdens of ageing and metabolic diseases. Adiponectin (ApN) is a hormone, mainly secreted by adipocytes, which exerts insulin-sensitizing and fat-burning properties in several tissues including the liver and the muscle. Its overexpression also increases lifespan in mice. In this study, we investigated whether an ApN receptor agonist, AdipoRon (AR), could slow muscle dysfunction, myosteatosis and degenerative muscle markers in middle-aged obese mice. The effects on myosteatosis were compared with those on NAFLD. METHODS: Three groups of mice were studied up to 62 weeks of age: One group received normal diet (ND), another, high-fat diet (HFD); and the last, HFD combined with AR given orally for almost 1 year. An additional group of young mice under an ND was used. Treadmill tests and micro-computed tomography (CT) were carried out in vivo. Histological, biochemical and molecular analyses were performed on tissues ex vivo. Bodipy staining was used to assess intramyocellular lipid (IMCL) and lipid droplet morphology. RESULTS: AR did not markedly alter diet-induced obesity. Yet, this treatment rescued exercise endurance in obese mice (up to 2.4-fold, P < 0.05), an event that preceded the improvement of insulin sensitivity. Dorsal muscles and liver densities, measured by CT, were reduced in obese mice (-42% and -109%, respectively, P < 0.0001), suggesting fatty infiltration. This reduction tended to be attenuated by AR. Accordingly, AR significantly mitigated steatosis and cellular ballooning at liver histology, thereby decreasing the NALFD activity score (-30%, P < 0.05). AR also strikingly reversed IMCL accumulation either due to ageing in oxidative fibres (types 1/2a, soleus) or to HFD in glycolytic ones (types 2x/2b, extensor digitorum longus) (-50% to -85%, P < 0.05 or less). Size of subsarcolemmal lipid droplets, known to be associated with adverse metabolic outcomes, was reduced as well. Alleviation of myosteatosis resulted from improved mitochondrial function and lipid oxidation. Meanwhile, AR halved aged-related accumulation of dysfunctional proteins identified as tubular aggregates and cylindrical spirals by electron microscopy (P < 0.05). CONCLUSIONS: Long-term AdipoRon treatment promotes 'healthy ageing' in obese middle-aged mice by enhancing endurance and protecting skeletal muscle and liver against the adverse metabolic and degenerative effects of ageing and caloric excess.

Inhibiting the inflammasome with MCC950 counteracts muscle pyroptosis and improves Duchenne muscular dystrophy.

Background: Duchenne muscular dystrophy (DMD) is the most common inherited human myopathy. Typically, the secondary process involving severe inflammation and necrosis exacerbate disease progression. Previously, we reported that the NLRP3 inflammasome complex plays a crucial role in this disorder. Moreover, pyroptosis, a form of programmed necrotic cell death, is triggered by NLRP3 via gasdermin D (GSDMD). So far, pyroptosis has never been described either in healthy muscle or in dystrophic muscle. The aim of this study was to unravel the role of NLRP3 inflammasome in DMD and explore a potentially promising treatment with MCC950 that selectively inhibits NLRP3. Methods: Four-week-old mdx mice (n=6 per group) were orally treated for 2 months with MCC950 (mdx-T), a highly potent, specific, small-molecule inhibitor of NLRP3, and compared with untreated (mdx) and wild-type (WT) mice. In vivo functional tests were carried out to measure the global force and endurance of mice. Ex vivo biochemical and molecular analyses were performed to evaluate the pathophysiology of the skeletal muscle. Finally, in vitro tests were conducted on primary cultures of DMD human myotubes. Results: After MCC950 treatment, mdx mice exhibited a significant reduction of inflammation, macrophage infiltration and oxidative stress (-20 to -65%, P<0.05 vs untreated mdx). Mdx-T mice displayed considerably less myonecrosis (-54%, P<0.05 vs mdx) and fibrosis (-75%, P<0.01 vs mdx). Moreover, a more mature myofibre phenotype, characterized by larger-sized fibres and higher expression of mature myosin heavy chains 1 and 7 was observed. Mdx-T also exhibited enhanced force and resistance to fatigue (+20 to 60%, P<0.05 or less). These beneficial effects resulted from MCC950 inhibition of both active caspase-1 (-46%, P=0.075) and cleaved gasdermin D (N-GSDMD) (-42% in medium-sized-fibres, P<0.001). Finally, the anti-inflammatory action and the anti-pyroptotic effect of MCC950 were also recapitulated in DMD human myotubes. Conclusion: Specific inhibition of the NLRP3 inflammasome can significantly attenuate the dystrophic phenotype. A novel finding of this study is the overactivation of GSDMD, which is hampered by MCC950. This ultimately leads to less inflammation and pyroptosis and to a better muscle maturation and function. Targeting NLRP3 might lead to an effective therapeutic approach for a better management of DMD.

Histological Methods to Assess Skeletal Muscle Degeneration and Regeneration in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive disease caused by the loss of function of the protein dystrophin. This protein contributes to the stabilisation of striated cells during contraction, as it anchors the cytoskeleton with components of the extracellular matrix through the dystrophin-associated protein complex (DAPC). Moreover, absence of the functional protein affects the expression and function of proteins within the DAPC, leading to molecular events responsible for myofibre damage, muscle weakening, disability and, eventually, premature death. Presently, there is no cure for DMD, but different treatments help manage some of the symptoms. Advances in genetic and exon-skipping therapies are the most promising intervention, the safety and efficiency of which are tested in animal models. In addition to in vivo functional tests, ex vivo molecular evaluation aids assess to what extent the therapy has contributed to the regenerative process. In this regard, the later advances in microscopy and image acquisition systems and the current expansion of antibodies for immunohistological evaluation together with the development of different spectrum fluorescent dyes have made histology a crucial tool. Nevertheless, the complexity of the molecular events that take place in dystrophic muscles, together with the rise of a multitude of markers for each of the phases of the process, makes the histological assessment a challenging task. Therefore, here, we summarise and explain the rationale behind different histological techniques used in the literature to assess degeneration and regeneration in the field of dystrophinopathies, focusing especially on those related to DMD.

Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle.

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative prognostic factor for several other disorders such as cardiovascular disease and cancer. Excessive lipid infiltration decreases muscle mass and strength. It also results in lipotoxicity and insulin resistance depending on total intramyocellular lipid content, lipid droplet (LD) morphology, and subcellular distribution. Fiber type (oxidative vs glycolytic) is also important, since oxidative fibers have a greater capacity to utilize lipids. Because of their crucial implications in pathophysiology, in-depth studies on LD dynamics and function in a fiber type-specific manner are warranted. Herein, a complete protocol is presented for the quantification of intramyocellular lipid content and analysis of LD morphology and subcellular distribution in a fiber type-specific manner. To this end, serial muscle cryosections were stained with the fluorescent dye Bodipy and antibodies against myosin heavy chain isoforms. This protocol enables the simultaneous processing of different muscles, saving time and avoiding possible artifacts and, thanks to a personalized macro created in Fiji, the automatization of LD analysis is also possible.

Walking down Skeletal Muscle Lane: From Inflammasome to Disease.

Over the last decade, innate immune system receptors and sensors called inflammasomes have been identified to play key pathological roles in the development and progression of numerous diseases. Among them, the nucleotide-binding oligomerization domain (NOD-), leucine-rich repeat (LRR-) and pyrin domain-containing protein 3 (NLRP3) inflammasome is probably the best characterized. To date, NLRP3 has been extensively studied in the heart, where its effects and actions have been broadly documented in numerous cardiovascular diseases. However, little is still known about NLRP3 implications in muscle disorders affecting non-cardiac muscles. In this review, we summarize and present the current knowledge regarding the function of NLRP3 in diseased skeletal muscle, and discuss the potential therapeutic options targeting the NLRP3 inflammasome in muscle disorders.

Adiponectin and Its Mimics on Skeletal Muscle: Insulin Sensitizers, Fat Burners, Exercise Mimickers, Muscling Pills or Everything Together?

Adiponectin (ApN) is a hormone abundantly secreted by adipocytes and it is known to be tightly linked to the metabolic syndrome. It promotes insulin-sensitizing, fat-burning, and anti-atherosclerotic actions, thereby effectively counteracting several metabolic disorders, including type 2 diabetes, obesity, and cardiovascular diseases. ApN is also known today to possess powerful anti-inflammatory/oxidative and pro-myogenic effects on skeletal muscles exposed to acute or chronic inflammation and injury, mainly through AdipoR1 (ApN specific muscle receptor) and AMP-activated protein kinase (AMPK) pathway, but also via T-cadherin. In this review, we will report all the beneficial and protective properties that ApN can exert, specifically on the skeletal muscle as a target tissue. We will highlight its effects and mechanisms of action, first in healthy skeletal muscle including exercised muscle, and second in diseased muscle from a variety of pathological conditions. In the end, we will go over some of AdipoRs agonists that can be easily produced and administered, and which can greatly mimic ApN. These interesting and newly identified molecules could pave the way towards future therapeutic approaches to potentially prevent or combat not only skeletal muscle disorders but also a plethora of other diseases with sterile inflammation or metabolic dysfunction.

AdipoRon, a new therapeutic prospect for Duchenne muscular dystrophy.

BACKGROUND: Adiponectin (ApN) is a hormone known to exhibit insulin-sensitizing, fat-burning, and anti-inflammatory properties in several tissues, including the skeletal muscle. Duchenne muscular dystrophy (DMD) is a devastating disease characterized by dystrophin deficiency with subsequent chronic inflammation, myofiber necrosis, and impaired regeneration. Previously, we showed that transgenic up-regulation of ApN could significantly attenuate the dystrophic phenotype in mdx mice (model of DMD). Recently, an orally active ApN receptor agonist, AdipoRon, has been identified. This synthetic small molecule has the advantage of being more easily produced and administrable than ApN. The aim of this study was to investigate the potential effects of AdipoRon on the dystrophic muscle. METHODS: Four-week-old mdx mice (n = 6-9 per group) were orally treated with AdipoRon (mdx-AR) for 8 weeks and compared with untreated (mdx) mice and to control (wild-type) mice. In vivo functional tests were carried out to measure the global force and endurance of mice. Ex vivo biochemical and molecular analyses were performed to evaluate the pathophysiology of the skeletal muscle. Finally, in vitro tests were conducted on primary cultures of healthy and DMD human myotubes. RESULTS: AdipoRon treatment mitigated oxidative stress (-30% to 45% for 4-hydroxy-2-nonenal and peroxiredoxin 3, P < 0.0001) as well as inflammation in muscles of mdx mice (-35% to 65% for interleukin 1 beta, tumour necrosis factor alpha, and cluster of differentiation 68, a macrophage maker, P < 0.0001) while increasing the anti-inflammatory cytokine, interleukin 10 (~5-fold, P < 0.0001). AdipoRon also improved the myogenic programme as assessed by a ~2-fold rise in markers of muscle proliferation and differentiation (P < 0.01 or less vs. untreated mdx). Plasma lactate dehydrogenase and creatine kinase were reduced by 30-40% in mdx-AR mice, reflecting less sarcolemmal damage (P < 0.0001). When compared with untreated mdx mice, mdx-AR mice exhibited enhanced physical performance with an increase in both muscle force and endurance and a striking restoration of the running capacity during eccentric exercise. AdipoRon mainly acted through ApN receptor 1 by increasing AMP-activated protein kinase signalling, which led to repression of nuclear factor-kappa B, up-regulation of utrophin (a dystrophin analogue), and a switch towards an oxidative and more resistant fibre phenotype. The effects of AdipoRon were then recapitulated in human DMD myotubes. CONCLUSIONS: These results demonstrate that AdipoRon exerts several beneficial effects on the dystrophic muscle. This molecule could offer promising therapeutic prospect for managing DMD or other muscle and inflammatory disorders.