Retinoic Acid Receptor Agonists Suppress Muscle Fatty Infiltration in Mice.

BACKGROUND: The infiltration of fat tissue into skeletal muscle, a condition referred to as muscle fatty infiltration or fatty degeneration, is regarded as an irreversible event that significantly compromises the motor function of skeletal muscle. PURPOSE: To investigate the effect of retinoic acid receptor (RAR) agonists in suppressing the adipogenic differentiation of fibroadipogenic progenitors (FAPs) in vitro and fatty infiltration after rotator cuff tear in mice. STUDY DESIGN: Controlled laboratory study. METHODS: FAPs isolated from mouse skeletal muscle were cultured in adipogenic differentiation medium in the presence or absence of an RAR agonist. At the end of cell culture, adipogenic differentiation was evaluated by gene expression analysis and oil red O staining. A mouse model of fatty infiltration-which includes the resection of the rotator cuff, removal of the humeral head, and denervation the supraspinatus muscle-was used to induce fatty infiltration in the supraspinatus muscle. The mice were orally or intramuscularly administered with an RAR agonist after the surgery. Muscle fatty infiltration was evaluated by histology and gene expression analysis. RESULTS: RAR agonists effectively inhibited the adipogenic differentiation of FAPs in vitro. Oral and intramuscular administration of RAR agonists suppressed the development of muscle fatty infiltration in the mice after rotator cuff tear. In accordance, we found a significant decrease in the number of intramuscular fat cells and suppressed expression in adipogenic markers. RAR agonists also increased the expression of the transcripts for collagens; however, an accumulation of collagenous tissues was not histologically evident in the present model. CONCLUSION: Muscle fatty infiltration can be alleviated by RAR agonists through suppressing the adipogenic differentiation of FAPs. The results also suggest that RAR agonists are potential therapeutic agents for treating patients who are at risk of developing muscle fatty infiltration. The consequence of the increased expression of collagen transcripts by RAR agonists needs to be clarified. CLINICAL RELEVANCE: RAR agonists can be used to prevent the development of muscle fatty infiltration after rotator cuff tear. Nevertheless, further studies are mandatory in a large animal model to examine the safety and efficacy of intramuscular injection of RAR agonists.

Granulocyte-colony stimulating factor enhances load-induced muscle hypertrophy in mice.

Granulocyte-colony stimulating factor (G-CSF) is a cytokine crucially involved in the regulation of granulopoiesis and the mobilization of hematopoietic stem cells from bone marrow. However, emerging data suggest that G-CSF exhibits more diverse functions than initially expected, such as conferring protection against apoptosis to neural cells and stimulating mitogenesis in cardiomyocytes and skeletal muscle stem cells after injury. In the present study, we sought to investigate the potential contribution of G-CSF to the regulation of muscle volume. We found that the administration of G-CSF significantly enhances muscle hypertrophy in two different muscle overload models. Interestingly, there was a significant increase in the transcripts of both G-CSF and G-CSF receptors in the muscles that were under overload stress. Using mutant mice lacking the G-CSF receptor, we confirmed that the anabolic effect is dependent on the G-CSF receptor signaling. Furthermore, we found that G-CSF increases the diameter of myotubes in vitro and induces the phosphorylation of AKT, mTOR, and ERK1/2 in the myoblast-like cell line C2C12 after differentiation induction. These findings indicate that G-CSF is involved in load-induced muscle hypertrophy and suggest that G-CSF is a potential agent for treating patients with muscle loss and sarcopenia.

Targeted ablation of p38α MAPK suppresses denervation-induced muscle atrophy.

The loss of skeletal muscle mass is a major cause of falls and fractures in the elderly, leading to compromised independence and a decrease in the quality of life. However, only a few therapeutic interventions leading to marginal clinical benefits in patients with this condition are currently available. Therefore, the demand to further understand the pathology of muscle atrophy and establish a treatment modality for patients with muscle atrophy is significant. p38α mitogen-activated protein kinase (p38α MAPK) is a ubiquitous signaling molecule that is implicated in various cellular functions, including cell proliferation, differentiation, and senescence. In the present study, we generated a mutant line in which p38α MAPK is specifically abrogated in muscle tissues. Compared with the control mice, these mutant mice are significantly resistant to denervation-induced muscle atrophy, suggesting that p38α MAPK positively regulates muscle atrophy. We also identified CAMK2B as a potential downstream target of p38α MAPK and found that the pharmacological inhibition of CAMK2B activity suppresses denervation-induced muscle atrophy. Altogether, our findings identify p38α MAPK as a novel regulator of muscle atrophy and suggest that the suppression of intracellular signaling mediated by p38α MAPK serves as a potential target for the treatment of muscle atrophy.

Antihyperalgesic effect of buprenorphine involves nociceptin/orphanin FQ peptide-receptor activation in rats with spinal nerve injury-induced neuropathy.

We evaluated the effect of buprenorphine, a mixed agonist for µ-opioid receptors and nociceptin/orphanin FQ peptide (NOP) receptors, in neuropathic rats, using the paw pressure test. Buprenorphine, administered i.p. at 50, but not 20, µg/kg, exhibited naloxone-reversible analgesic activity in naïve rats. In contrast, buprenorphine at 0.5 - 20 µg/kg produced a naloxonesensitive antihyperalgesic effect in the L5 spinal nerve-injured neuropathic rats. Intrathecal injection of [N-Phe(1)]nociceptin(1-13)NH2, a NOP-receptor antagonist, reversed the effect of buprenorphine in neuropathic rats, but not in naïve rats. Together, buprenorphine suppresses neuropathic hyperalgesia by activating NOP and opioid receptors, suggesting its therapeutic usefulness in treatment of neuropathic pain.

Hydrogen sulfide-induced mechanical hyperalgesia and allodynia require activation of both Cav3.2 and TRPA1 channels in mice.

BACKGROUND AND PURPOSE: Hydrogen sulfide, a gasotransmitter, facilitates somatic pain signals via activation of Ca(v)3.2 T-type calcium channels in rats. Given evidence for the activation of transient receptor potential ankyrin-1 (TRPA1) channels by H(2)S, we asked whether TRPA1 channels, in addition to Ca(v)3.2 channels, contribute to the H(2)S-induced mechanical hyperalgesia and allodynia in mice. EXPERIMENTAL APPROACH: Mechanical hyperalgesia and allodynia were evaluated by the von Frey test in mice. Ca(v)3.2 or TRPA1 channels in the sensory neurons were silenced by repeated intrathecal administration of antisense oligodeoxynucleotides in mice. KEY RESULTS: Intraplantar administration of NaHS evoked hyperalgesia and allodynia in mice, an effect attenuated or abolished by NNC 55-0396 or mibefradil, T-type calcium channel blockers, and by ascorbic acid or zinc chloride, known to selectively inhibit Ca(v)3.2 channels, out of the three isoforms of T-type calcium channels. Silencing of Ca(v)3.2 channels in the sensory neurons also prevented the NaHS-induced hyperalgesia and allodynia in mice. The NaHS-induced hyperalgesia and allodynia in mice were significantly suppressed by AP18, a TRPA1 channel blocker, and by silencing of TRPA1 channels in the sensory neurons. CONCLUSIONS AND IMPLICATIONS: Mechanical hyperalgesia and allodynia induced by NaHS/H(2)S required activation of both Ca(v)3.2 and TRPA1 channels in mice.

Topical application of disodium isostearyl 2-O-L-ascorbyl phosphate, an amphiphilic ascorbic acid derivative, reduces neuropathic hyperalgesia in rats.

BACKGROUND AND PURPOSE: Ca(v) 3.2 T-type calcium channels, targeted by H(2) S, are involved in neuropathic hyperalgesia in rats and ascorbic acid inhibits Ca(v) 3.2 channels. Therefore, we evaluated the effects of intraplantar (i.pl.) administration of ascorbic acid or topical application of disodium isostearyl 2-O-L-ascorbyl phosphate (DI-VCP), a skin-permeable ascorbate derivative on hyperalgesia induced by NaHS, an H(2) S donor, and on neuropathic hyperalgesia. EXPERIMENTAL APPROACH: In rats mechanical hyperalgesia was evoked by i.pl. NaHS, and neuropathic hyperalgesia was induced by L5 spinal nerve cutting (L5SNC) or by repeated administration of paclitaxel, an anti-cancer drug. Dermal ascorbic acid levels were determined colorimetrically. KEY RESULTS: The NaHS-evoked Ca(v) 3.2 channel-dependent hyperalgesia was inhibited by co-administered ascorbic acid. Topical application of DI-VCP, but not ascorbic acid, prevented the NaHS-evoked hyperalgesia, and also increased dermal ascorbic acid levels. Neuropathic hyperalgesia induced by L5SNC or paclitaxel was reversed by i.pl. NNC 55-0396, a selective T-type calcium channel blocker, ascorbic acid or DI-VCP, and by topical DI-VCP, but not by topical ascorbic acid. The effects of i.pl. ascorbic acid and topical DI-VCP in the paclitaxel-treated rats were characterized by the faster onset and greater magnitude, compared with their effects in the L5SNC rats. Dermal ascorbic acid levels in the hindpaw significantly decreased after paclitaxel treatment, but not L5SNC, which was reversed by topical DI-VCP. CONCLUSIONS AND IMPLICATIONS: Ascorbic acid, known to inhibit Ca(v) 3.2 channels, suppressed neuropathic hyperalgesia. DI-VCP ointment for topical application may be of benefit in the treatment of neuropathic pain.

Upregulation of Ca(v)3.2 T-type calcium channels targeted by endogenous hydrogen sulfide contributes to maintenance of neuropathic pain.

Hydrogen sulfide (H(2)S) formed from l-cysteine by multiple enzymes including cystathionine-gamma-lyase (CSE) is now considered a gasotransmitter in the mammalian body. Our previous studies have shown that H(2)S activates/sensitizes Ca(v)3.2 T-type Ca(2+) channels, leading to facilitation of somatic and visceral nociception, and that CSE-derived endogenous H(2)S participates in inflammatory pain. Here, we show novel evidence for involvement of the endogenous H(2)S-Ca(v)3.2 pathway in neuropathic pain. In the rat subjected to the right L5 spinal nerve cutting (L5SNC), a neuropathic pain model, i.p. administration of dl-propargylglycine (PPG) and beta-cyanoalanine, irreversible and reversible CSE inhibitors, respectively, strongly suppressed the neuropathic hyperalgesia/allodynia. The anti-hyperalgesic effect of PPG was reversed by intraplantar administration of NaHS, a donor for H(2)S, in the L5SNC rat. Intraplantar administration or topical application of mibefradil, a T-type Ca(2+) channel blocker, reversed hyperalgesia in the L5SNC rat. The protein levels of Ca(v)3.2, but not CSE, in the ipsilateral L4, L5 and L6 dorsal root ganglia were dramatically upregulated in the L5SNC rat. Finally, silencing of Ca(v)3.2 in DRG by repeated intrathecal administration of Ca(v)3.2-targeting siRNA significantly attenuated the neuropathic hyperalgesia in the L5SNC rat. In conclusion, our data suggest that Ca(v)3.2 T-type Ca(2+) channels in sensory neurons are upregulated and activated/sensitized by CSE-derived endogenous H(2)S after spinal nerve injury, contributing to the maintenance of neuropathic pain. We thus propose that Ca(v)3.2 and CSE could be targets for the development of therapeutic drugs for the treatment of neuropathic pain.