Abstract
Hyaluronan (HA) is a nonsulfated glycosaminoglycan that has been widely used for biomedical applications. Here, we have analyzed the effect of HA on the rescue of primary cells under stress as well as its potential to recover muscle atrophy and validated the developed model in vitro using primary muscle cells derived from rats. The potentials of different HAs were elucidated through comparative analyses using pharmaceutical grade a) high (HHA) and b) low molecular weight (LHA) hyaluronans, c) hybrid cooperative complexes (HCC) of HA in three experimental set-ups. The cells were characterized based on the expression of myogenin, a muscle-specific biomarker, and the proliferation was analyzed using Time-Lapse Video Microscopy (TLVM). Cell viability in response to H2O2 challenge was evaluated by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the expression of the superoxide dismutase enzyme (SOD-2) was assessed by western blotting. Additionally, in order to establish an in vitro model of atrophy, muscle cells were treated with tumor necrosis factor-alpha (TNF-α), along with hyaluronans. The expression of Atrogin, MuRF-1, nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-kB), and Forkhead-box-(Fox)-O-3 (FoxO3a) was evaluated by western blotting to elucidate the molecular mechanism of atrophy. The results showed that HCC and HHA increased cell proliferation by 1.15 and 2.3 folds in comparison to un-treated cells (control), respectively. Moreover, both pre- and post-treatments of HAs restored the cell viability, and the SOD-2 expression was found to be reduced by 1.5 fold in HA-treated cells as compared to the stressed condition. Specifically in atrophic stressed cells, HCC revealed a noteworthy beneficial effect on the myogenic biomarkers indicating that it could be used as a promising platform for tissue regeneration with specific attention to muscle cell protection against stressful agents.
Highlights
Diverse physiological and pathological conditions such as inactivity, aging, starvation, diabetes, cachexia, and cancer can cause reduced synthesis and increased breakdown of muscle proteins, leading to lessened muscle mass, known as muscle atrophy [1, 2]
The results demonstrated that hybrid cooperative complexes (HCC) promoted cell proliferation faster than LHA and high molecular weight hyaluronan (HHA) after 24 h of treatment
This study demonstrated that hyaluronan and, in particular, hybrid cooperative complexes made by high and low molecular weight chains of HA inhibited or counteracted the biochemical pathways involved in cell stress and damage preserving viability, phenotype, and overall functionality
Summary
Diverse physiological and pathological conditions such as inactivity, aging (i.e., age-related sarcopenia), starvation, diabetes, cachexia, and cancer can cause reduced synthesis and increased breakdown of muscle proteins, leading to lessened muscle mass, known as muscle atrophy [1, 2]. Skeletal muscle atrophy is an important clinical disorder mediated by the activation of proteolytic systems inducing muscle weakness and mass reduction [3]. Atrophy resulted from an increase in protein degradation, loss of muscle mass [6, 1], and a reduction of protein synthesis (Fig 1). This process is predominantly regulated by myogenic transcription factors, the atrogenes, including FoxO3a (Forkhead box (Fox)-O 3), atrogin, known as MAFbx, muscle-specific ring finger protein (MuRF-1) [6], and myogenic regulatory protein such as myogenin and desmin [7], and these factors are used as biomarkers of muscle functionality [8]. The specific molecular mechanisms underlying the cell damage have not been fully
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