Alterations in SAMD9, AHSG, FRG2C, and FGFR4 Genes in a Case of Late-Onset Massive Tumoral Calcinosis.

Background/Objective: Tumoral calcinosis (TC) is a rare, arcane, and debilitating disorder of phosphate metabolism manifesting as hard masses in soft tissues. Primary hyperphosphatemic TC has been shown to be caused by pathogenic variants in the genes encoding FGF23, GALNT3, and KLOTHO. We report a case of massive TC mechanistically associated with phosphatonin resistance associated with heterozygous alterations in the sterile alfa motif domain-containing protein-9 gene (SAMD9), alfa 2-Heremans-Schmid glycoprotein gene (AHSG), FSHD region gene 2-family member-C gene (FRG2C), and fibroblast growth factor receptor-4 gene (FGFR4). Case Report: A middle-aged Malay woman with systemic sclerosis presented with painful hard lumps of her axillae, lower limbs, and external genitalia. She was eucalcemic with mild hyperphosphatemia associated with reduced urinary phosphate excretion. Magnetic resonance imaging revealed calcified soft tissue masses. Paradoxically, the serum intact FGF23 level increased to 89.6 pg/mL, corroborated by Western blots, which also showed overexpression of sFRP4 and MEPE, consistent with phosphatonin resistance. Discussion: Whole genome sequencing identified 2 heterozygous alterations (p.A454T and p.T479M) in SAMD9, 2 heterozygous alterations (p.M248T and p.S256T) in AHSG, a frameshift alteration (p.Arg156fs) in FRG2C, and a heterozygous alteration (p.G388R) in FGFR4, all of which are associated with calcinosis. Nonsynonymous alterations of FRP4 and MEPE were also detected. Conclusion: This highlights that the simultaneous occurrence of alterations in several genes critical in phosphate homeostasis may trigger massive TC despite their heterozygosity. These findings should prompt functional studies in cell and animal models to reveal mechanistic insights in the pathogenesis of such crippling mineralization disorders.

Immune checkpoint inhibitors in HCC: Cellular, molecular and systemic data.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer related deaths in the world, and for patients with advanced disease there are few therapeutic options available. The complex immunological microenvironment of HCC and the success of immunotherapy in several types of tumours, has raised the prospect of potential benefit for immune based therapies, such as immune checkpoint inhibitors (ICIs), in HCC. This has led to significant breakthrough research, numerous clinical trials and the rapid approval of multiple systemic drugs for HCC by regulatory bodies worldwide. Although some patients responded well to ICIs, many have failed to achieve significant benefit, while others showed unexpected and paradoxical deterioration. The aim of this review is to discuss the pathophysiology of HCC, the tumour microenvironment, key clinical trials evaluating ICIs in HCC, various resistance mechanisms to ICIs, and possible ways to overcome these impediments to improve patient outcomes.

Cell adhesion an important determinant of myogenesis and satellite cell activity.

Skeletal muscles represent a complex and highly organised tissue responsible for all voluntary body movements. Developed through an intricate and tightly controlled process known as myogenesis, muscles form early in development and are maintained throughout life. Due to the constant stresses that muscles are subjected to, skeletal muscles maintain a complex course of regeneration to both replace and repair damaged myofibers and to form new functional myofibers. This process, made possible by a pool of resident muscle stem cells, termed satellite cells, and controlled by an array of transcription factors, is additionally reliant on a diverse range of cell adhesion molecules and the numerous signaling cascades that they initiate. This article will review the literature surrounding adhesion molecules and their roles in skeletal muscle myogenesis and repair.

Targeting the PI3K/Akt/mTOR Pathway in Hepatocellular Carcinoma.

Despite advances in the treatment of cancers through surgical procedures and new pharmaceuticals, the treatment of hepatocellular carcinoma (HCC) remains challenging as reflected by low survival rates. The PI3K/Akt/mTOR pathway is an important signaling mechanism that regulates the cell cycle, proliferation, apoptosis, and metabolism. Importantly, deregulation of the PI3K/Akt/mTOR pathway leading to activation is common in HCC and is hence the subject of intense investigation and the focus of current therapeutics. In this review article, we consider the role of this pathway in the pathogenesis of HCC, focusing on its downstream effectors such as glycogen synthase kinase-3 (GSK-3), cAMP-response element-binding protein (CREB), forkhead box O protein (FOXO), murine double minute 2 (MDM2), p53, and nuclear factor-κB (NF-κB), and the cellular processes of lipogenesis and autophagy. In addition, we provide an update on the current ongoing clinical development of agents targeting this pathway for HCC treatments.

Are Interactions between Epicardial Adipose Tissue, Cardiac Fibroblasts and Cardiac Myocytes Instrumental in Atrial Fibrosis and Atrial Fibrillation?

Atrial fibrillation is very common among the elderly and/or obese. While myocardial fibrosis is associated with atrial fibrillation, the exact mechanisms within atrial myocytes and surrounding non-myocytes are not fully understood. This review considers the potential roles of myocardial fibroblasts and myofibroblasts in fibrosis and modulating myocyte electrophysiology through electrotonic interactions. Coupling with (myo)fibroblasts in vitro and in silico prolonged myocyte action potential duration and caused resting depolarization; an optogenetic study has verified in vivo that fibroblasts depolarized when coupled myocytes produced action potentials. This review also introduces another non-myocyte which may modulate both myocardial (myo)fibroblasts and myocytes: epicardial adipose tissue. Epicardial adipocytes are in intimate contact with myocytes and (myo)fibroblasts and may infiltrate the myocardium. Adipocytes secrete numerous adipokines which modulate (myo)fibroblast and myocyte physiology. These adipokines are protective in healthy hearts, preventing inflammation and fibrosis. However, adipokines secreted from adipocytes may switch to pro-inflammatory and pro-fibrotic, associated with reactive oxygen species generation. Pro-fibrotic adipokines stimulate myofibroblast differentiation, causing pronounced fibrosis in the epicardial adipose tissue and the myocardium. Adipose tissue also influences myocyte electrophysiology, via the adipokines and/or through electrotonic interactions. Deeper understanding of the interactions between myocytes and non-myocytes is important to understand and manage atrial fibrillation.

Paraneoplastic Secretion of Multiple Phosphatonins From a Deep Fibrous Histiocytoma Causing Oncogenic Osteomalacia.

CONTEXT: Literature suggests that oncogenic osteomalacia is usually caused by a benign mesenchymal tumor secreting fibroblast growth factor subtype-23 (FGF-23), but the involvement of other phosphatonins has only been scarcely reported. We have previously published a seemingly typical case of oncogenic osteomalacia. Following curative neoplasm resection, we now report unique molecular characteristics and biology of this tumor. CASE DESCRIPTION: A 25-year-old man had been diagnosed with severe oncogenic osteomalacia that gradually crippled him over 6 years. 68Ga-DOTA-TATE positron emission tomography/computed tomography scan localized the culprit tumor to his left sole, which on resection revealed a deep fibrous histiocytoma displaying a proliferation of spindle cells with storiform pattern associated with multinucleated giant cells resembling osteoclasts. Circulating FGF-23, which was elevated more than 2-fold, declined to undetectable levels 24 h after surgery. Microarray analysis revealed increased tumor gene expression of the phosphatonins FGF-23, matrix extracellular phosphoglycoprotein (MEPE) and secreted frizzled-related protein subtype 4, with elevated levels of all 3 proteins confirmed through immunoblot analysis. Differential expression of genes involved in bone formation and bone mineralization were further identified. The patient made an astonishing recovery from being wheelchair bound to fully self-ambulant 2 months postoperatively. CONCLUSION: This report describes oncogenic osteomalacia due to a deep fibrous histiocytoma, which coincidentally has been found to induce profound muscle weakness via the overexpression of 3 phosphatonins, which resolved fully upon radical resection of the tumor. Additionally, genes involved in bone formation and bone remodeling contribute to the molecular signature of oncogenic osteomalacia.

Mitochondrial oxidative capacity and NAD+ biosynthesis are reduced in human sarcopenia across ethnicities.

The causes of impaired skeletal muscle mass and strength during aging are well-studied in healthy populations. Less is known on pathological age-related muscle wasting and weakness termed sarcopenia, which directly impacts physical autonomy and survival. Here, we compare genome-wide transcriptional changes of sarcopenia versus age-matched controls in muscle biopsies from 119 older men from Singapore, Hertfordshire UK and Jamaica. Individuals with sarcopenia reproducibly demonstrate a prominent transcriptional signature of mitochondrial bioenergetic dysfunction in skeletal muscle, with low PGC-1α/ERRα signalling, and downregulation of oxidative phosphorylation and mitochondrial proteostasis genes. These changes translate functionally into fewer mitochondria, reduced mitochondrial respiratory complex expression and activity, and low NAD+ levels through perturbed NAD+ biosynthesis and salvage in sarcopenic muscle. We provide an integrated molecular profile of human sarcopenia across ethnicities, demonstrating a fundamental role of altered mitochondrial metabolism in the pathological loss of skeletal muscle mass and function in older people.

G protein-coupled receptor kinase 2 regulates mitochondrial bioenergetics and impairs myostatin-mediated autophagy in muscle cells.

G protein-coupled receptor kinase 2 (GRK2) is an important protein involved in ß-adrenergic receptor desensitization. In addition, studies have shown GRK2 can modulate different metabolic processes in the cell. For instance, GRK2 has been recently shown to promote mitochondrial biogenesis and increase ATP production. However, the role of GRK2 in skeletal muscle and the signaling mechanisms that regulate GRK2 remain poorly understood. Myostatin is a well-known myokine that has been shown to impair mitochondria function. Here, we have assessed the role of myostatin in regulating GRK2 and the subsequent downstream effect of myostatin regulation of GRK2 on mitochondrial respiration in skeletal muscle. Myostatin treatment promoted the loss of GRK2 protein in myoblasts and myotubes in a time- and dose-dependent manner, which we suggest was through enhanced ubiquitin-mediated protein loss, as treatment with proteasome inhibitors partially rescued myostatin-mediated loss of GRK2 protein. To evaluate the effects of GRK2 on mitochondrial respiration, we generated stable myoblast lines that overexpress GRK2. Stable overexpression of GRK2 resulted in increased mitochondrial content and enhanced mitochondrial/oxidative respiration. Interestingly, although overexpression of GRK2 was unable to prevent myostatin-mediated impairment of mitochondrial respiratory function, elevated levels of GRK2 blocked the increased autophagic flux observed following treatment with myostatin. Overall, our data suggest a novel role for GRK2 in regulating mitochondria mass and mitochondrial respiration in skeletal muscle.

Loss of Parkin impairs mitochondrial function and leads to muscle atrophy.

Parkinson's disease is a neurodegenerative disease characterized by tremors, muscle stiffness, and muscle weakness. Molecular genetic analysis has confirmed that mutations in PARKIN and PINK1 genes, which play major roles in mitochondrial quality control and mitophagy, are frequently associated with Parkinson's disease. PARKIN is an E3 ubiquitin ligase that translocates to mitochondria during loss of mitochondrial membrane potential to increase mitophagy. Although muscle dysfunction is noted in Parkinson's disease, little is known about the involvement of PARKIN in the muscle phenotype of Parkinson's disease. In this study, we report that the mitochondrial uncoupler CCCP promotes PINK1/PARKIN-mediated mitophagy in myogenic C2C12 cells. As a result of this excess mitophagy, we show that CCCP treatment of myotubes leads to the development of myotube atrophy in vitro. Surprisingly, we also found that siRNA-mediated knockdown of Parkin results in impaired mitochondrial turnover. In addition, knockdown of Parkin led to myotubular atrophy in vitro. Consistent with these in vitro results, Parkin knockout muscles showed impaired mitochondrial function and smaller myofiber area, suggesting that Parkin function is required for post-natal skeletal muscle growth and development.

Irisin treatment improves healing of dystrophic skeletal muscle.

BACKGROUND: Irisin is an exercise induced myokine that is shown to promote browning of adipose tissue and hence, increase energy expenditure. Furthermore, our unpublished results indicate that Irisin improves myogenic differentiation and induces skeletal muscle hypertrophy. Since exercise induced skeletal muscle hypertrophy improves muscle strength, we wanted to investigate if ectopic injection of Irisin peptide improves skeletal muscle function in a mouse model of muscular dystrophy. This utility of Irisin peptide is yet to be studied in animal models. METHODS: In order to test this hypothesis, we expressed and purified recombinant murine Irisin peptide from E. coli. Three- to six-week-old male mdx mice were injected IP with either vehicle (dialysis buffer) or Irisin recombinant peptide for two or four weeks, three times-a-week. RESULTS: Irisin injection increased muscle weights and enhanced grip strength in mdx mice. Improved muscle strength can be attributed to the significant hypertrophy observed in the Irisin injected mdx mice. Moreover, Irisin treatment resulted in reduced accumulation of fibrotic tissue and myofiber necrosis in mdx mice. In addition, Irisin improved sarcolemmal stability, which is severely compromised in mdx mice. CONCLUSION: Irisin injection induced skeletal muscle hypertrophy, improved muscle strength and reduced necrosis and fibrotic tissue in a murine dystrophy model. These results demonstrate the potential therapeutic value of Irisin in muscular dystrophy.

Irisin is a pro-myogenic factor that induces skeletal muscle hypertrophy and rescues denervation-induced atrophy.

Exercise induces expression of the myokine irisin, which is known to promote browning of white adipose tissue and has been shown to mediate beneficial effects following exercise. Here we show that irisin induces expression of a number of pro-myogenic and exercise response genes in myotubes. Irisin increases myogenic differentiation and myoblast fusion via activation of IL6 signaling. Injection of irisin in mice induces significant hypertrophy and enhances grip strength of uninjured muscle. Following skeletal muscle injury, irisin injection improves regeneration and induces hypertrophy. The effects of irisin on hypertrophy are due to activation of satellite cells and enhanced protein synthesis. In addition, irisin injection rescues loss of skeletal muscle mass following denervation by enhancing satellite cell activation and reducing protein degradation. These data suggest that irisin functions as a pro-myogenic factor in mice.

Narciclasine attenuates diet-induced obesity by promoting oxidative metabolism in skeletal muscle.

Obesity develops when caloric intake exceeds metabolic needs. Promoting energy expenditure represents an attractive approach in the prevention of this fast-spreading epidemic. Here, we report a novel pharmacological strategy in which a natural compound, narciclasine (ncls), attenuates diet-induced obesity (DIO) in mice by promoting energy expenditure. Moreover, ncls promotes fat clearance from peripheral metabolic tissues, improves blood metabolic parameters in DIO mice, and protects these mice from the loss of voluntary physical activity. Further investigation suggested that ncls achieves these beneficial effects by promoting a shift from glycolytic to oxidative muscle fibers in the DIO mice thereby enhancing mitochondrial respiration and fatty acid oxidation (FAO) in the skeletal muscle. Moreover, ncls strongly activates AMPK signaling specifically in the skeletal muscle. The beneficial effects of ncls treatment in fat clearance and AMPK activation were faithfully reproduced in vitro in cultured murine and human primary myotubes. Mechanistically, ncls increases cellular cAMP concentration and ADP/ATP ratio, which further lead to the activation of AMPK signaling. Blocking AMPK signaling through a specific inhibitor significantly reduces FAO in myotubes. Finally, ncls also enhances mitochondrial membrane potential and reduces the formation of reactive oxygen species in cultured myotubes.

The transcription factor SOX6 contributes to the developmental origins of obesity by promoting adipogenesis.

An association between impaired fetal growth and the postnatal development of obesity has been established. Here, by comparing adipocytes differentiated from mesenchymal stem cells (MSCs) taken from the umbilical cord and derived from normal and growth-restricted neonates, we identified the transcription factor SOX6 as highly expressed only in growth-restricted individuals. We found that SOX6 regulates adipogenesis in vertebrate species by activating adipogenic regulators including PPARγ, C/EBPα and MEST. We further show that SOX6 interacts with ß-catenin in adipocytes, suggesting an inhibition of WNT/ß-catenin signaling, thereby promoting adipogenesis. The upstream regulatory region of the MEST gene in MSCs from growth-restricted subjects harbors hypomethylated CpGs next to SOX6 binding motifs, and we found that SOX6 binding is impaired by adjacent CpG methylation. In summary, we report that SOX6 is a novel regulator of adipogenesis synergizing with epigenetic mechanisms.

Myostatin: expanding horizons.

Myostatin is a secreted growth and differentiation factor that belongs to the TGF-ß superfamily. Myostatin is predominantly synthesized and expressed in skeletal muscle and thus exerts a huge impact on muscle growth and function. In keeping with its negative role in myogenesis, myostatin expression is tightly regulated at several levels including epigenetic, transcriptional, post-transcriptional, and post-translational. New revelations regarding myostatin regulation also offer mechanisms that could be exploited for developing myostatin antagonists. Increasingly, it is becoming clearer that besides its conventional role in muscle, myostatin plays a critical role in metabolism. Hence, molecular mechanisms by which myostatin regulates several key metabolic processes need to be further explored.

Inactivation of PPARß/δ adversely affects satellite cells and reduces postnatal myogenesis.

Peroxisome proliferator-activated receptor ß/δ (PPARß/δ) is a ubiquitously expressed gene with higher levels observed in skeletal muscle. Recently, our laboratory showed (Bonala S, Lokireddy S, Arigela H, Teng S, Wahli W, Sharma M, McFarlane C, Kambadur R. J Biol Chem 287: 12935-12951, 2012) that PPARß/δ modulates myostatin activity to induce myogenesis in skeletal muscle. In the present study, we show that PPARß/δ-null mice display reduced body weight, skeletal muscle weight, and myofiber atrophy during postnatal development. In addition, a significant reduction in satellite cell number was observed in PPARß/δ-null mice, suggesting a role for PPARß/δ in muscle regeneration. To investigate this, tibialis anterior muscles were injured with notexin, and muscle regeneration was monitored on days 3, 5, 7, and 28 postinjury. Immunohistochemical analysis revealed an increased inflammatory response and reduced myoblast proliferation in regenerating muscle from PPARß/δ-null mice. Histological analysis confirmed that the regenerated muscle fibers of PPARß/δ-null mice maintained an atrophy phenotype with reduced numbers of centrally placed nuclei. Even though satellite cell numbers were reduced before injury, satellite cell self-renewal was found to be unaffected in PPARß/δ-null mice after regeneration. Previously, our laboratory had showed (Bonala S, Lokireddy S, Arigela H, Teng S, Wahli W, Sharma M, McFarlane C, Kambadur R. J Biol Chem 287: 12935-12951, 2012) that inactivation of PPARß/δ increases myostatin signaling and inhibits myogenesis. Our results here indeed confirm that inactivation of myostatin signaling rescues the atrophy phenotype and improves muscle fiber cross-sectional area in both uninjured and regenerated tibialis anterior muscle from PPARß/δ-null mice. Taken together, these data suggest that absence of PPARß/δ leads to loss of satellite cells, impaired skeletal muscle regeneration, and postnatal myogenesis. Furthermore, our results also demonstrate that functional antagonism of myostatin has utility in rescuing these effects.

Mega roles of microRNAs in regulation of skeletal muscle health and disease.

Skeletal muscle is a dynamic tissue with remarkable plasticity. Skeletal muscle growth and regeneration are highly organized processes thus it is not surprising that a high degree of complexity exists in the regulation of these processes. Recent discovery of non-coding microRNAs (miRNAs) has prompted extensive research in understanding the roles of these molecules in skeletal muscle. Research so far shows that miRNAs play a very significant role at every aspect of muscle biology. Besides muscle growth, development, and regeneration miRNAs are also involved in the pathology of muscle diseases and metabolism. In this review, recent advancements in miRNA function during myogenesis, exercise, atrophy, aging, and dystrophy are discussed.

Lack of myostatin reduces MyoD induced myogenic potential of primary muscle fibroblasts.

Conversion of skin fibroblasts into myoblasts by transducing the cells with myogenic master regulator MyoD has been in practice for more than two decades. The purpose of such conversion is due to scarcity of muscle biopsies during muscle wasting, hence conversion of fibroblasts to myogenic lineage from various genetic backgrounds offers a great alternative for cell therapies. Here, we have investigated if eliminating Myostatin, a potent negative regulator of myogenesis, could improve the myogenic conversion of fibroblasts. In the present study, we have isolated primary muscle fibroblasts from the skeletal muscles of wild-type (WT) and myostatin null (Mstn(-/-)) mice and transduced the muscle fibroblasts with MyoD using adenoviral, lentiviral transduction, and electroporation methods. In contrast to what we predicted, it is only in WT muscle fibroblasts we detected significant ectopic expression of MyoD, and myogenic conversion. Muscle fibroblasts from Mstn(-/-) genotype failed to take up as much MyoD using the three methods and, therefore, failed to form myotubes. The aforesaid condition of greater MyoD uptake by WT muscle fibroblasts was attributed to the presence of adenoviral receptors, which facilitated adenoviral transduction. However, in Mstn(-/-) fibroblasts we detected negligible levels of adenovirus receptors. Moreover, we also detected significantly higher levels of MyoD antagonists, c-Fos, c-Jun, and cyclin D1 in Mstn(-/-) muscle fibroblasts. Taken together, our results demonstrate that lack of myostatin reduces myogenic potential of muscle fibroblasts by inhibiting MyoD function.