Stine Juhl Petersson

Characterization of DLK1+ Cells Emerging During Skeletal Muscle Remodeling in Response to Myositis, Myopathies, and Acute Injury

Delta like 1 (DLK1) has been proposed to act as a regulator of cell fate determination and is linked to the development of various tissues including skeletal muscle. Herein we further investigated DLK1 expression during skeletal muscle remodeling. Although practically absent in normal adult muscle, DLK1 was upregulated in all human myopathies analyzed, including Duchenne‐ and Becker muscular dystrophies. Substantial numbers of DLK1+ satellite cells were observed in normal neonatal and Duchenne muscle, and furthermore, myogenic DLK1+ cells were identified during muscle regeneration in animal models in which the peak expression of Dlk1 mRNA and protein coincided with that of myoblast differentiation and fusion. In addition to perivascular DLK1+ cells, interstitial DLK1+ cells were numerous in regenerating muscle, and in agreement with colocalization studies of DLK1 and CD90/DDR2, qPCR of fluorescence‐activated cell sorting DLK1+ and DLK1− cells revealed that the majority of DLK1+ cells isolated at day 7 of regeneration had a fibroblast‐like phenotype. The existence of different DLK1+ populations was confirmed in cultures of primary derived myogenic cells, in which large flat nonmyogenic DLK1+ cells and small spindle‐shaped cells coexpressing DLK1 and muscle‐specific markers were observed. Myogenic differentiation was achieved when sorted DLK1+ cells were cocultured together with primary myoblasts revealing a myogenic potential that was 10% of the DLK1− population. Transplantation of DLK1+ cells into lacerated muscle did, however, not give rise to DLK1+ cell‐derived myofibers. We suggest that the DLK1+ subpopulations identified herein each may contribute at different levels/time points to the processes involved in muscle development and remodeling. STEM CELLS 2009;27:898–908

Markers of autophagy are adapted to hyperglycaemia in skeletal muscle in type 2 diabetes

Aims/hypothesisAutophagy is a catabolic process that maintains cellular homeostasis by degradation of protein aggregates and selective removal of damaged organelles, e.g. mitochondria (mitophagy). Insulin resistance in skeletal muscle has been linked to mitochondrial dysfunction and altered protein metabolism. Here, we investigated whether abnormalities in autophagy are present in human muscle in obesity and type 2 diabetes.MethodsUsing a case–control design, skeletal muscle biopsies obtained in the basal and insulin-stimulated states from patients with type 2 diabetes during both euglycaemia and hyperglycaemia, and from glucose-tolerant lean and obese individuals during euglycaemia, were used for analysis of mRNA levels, protein abundance and phosphorylation of autophagy-related proteins.ResultsMuscle transcript levels of autophagy-related genes (ULK1, BECN1, PIK3C3, ATG5, ATG7, ATG12, GABARAPL1, MAP1LC3B, SQSTM1, TP53INP2 and FOXO3A [also known as FOXO3]), including some specific for mitophagy (BNIP3, BNIP3L and MUL1), and protein abundance of autophagy-related gene (ATG)7 and Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), as well as content and phosphorylation of forkhead box O3A (FOXO3A) were similar among the groups. Insulin reduced lipidation of microtubule-associated protein light chain 3 (LC3)B-I to LC3B-II, a marker of autophagosome formation, with no effect on p62/sequestosome 1 (SQSTM1) content in muscle of lean and obese individuals. In diabetic patients, insulin action on LC3B was absent and p62/SQSTM1 content increased when studied under euglycaemia, whereas the responses of LC3B and p62/SQSTM1 to insulin were normalised during hyperglycaemia.Conclusions/interpretationOur results demonstrate that the levels of autophagy-related genes and proteins in muscle are normal in obesity and type 2 diabetes. This suggests that muscle autophagy in type 2 diabetes has adapted to hyperglycaemia, which may contribute to preserve muscle mass.

A PGC-1α- and muscle fibre type-related decrease in markers of mitochondrial oxidative metabolism in skeletal muscle of humans with inherited insulin resistance

Aims/hypothesisInsulin resistance in obesity and type 2 diabetes is related to abnormalities in mitochondrial oxidative phosphorylation (OxPhos) in skeletal muscle. We tested the hypothesis that mitochondrial oxidative metabolism is impaired in muscle of patients with inherited insulin resistance and defective insulin signalling.MethodsSkeletal muscle biopsies obtained from carriers (n = 6) of a mutation in the tyrosine kinase domain of the insulin receptor gene (INSR) and matched healthy controls (n = 15) were used for discovery-mode microarray-based transcriptional profiling combined with biological pathway analysis. Findings were validated by quantitative real-time PCR, immunoblotting and activity assays.ResultsIn INSR mutation carriers, insulin resistance was associated with a coordinated downregulation of OxPhos genes in skeletal muscle. This was related to a 46% decrease in mRNA levels (p = 0.036) of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and 25–50% lower protein content of OxPhos subunits encoded by mitochondrial (ND6, p = 0.042) and nuclear DNA (UQCRC1, p = 0.001; SDHA, p = 0.067; COX5A, p = 0.017 and ATP5B, p = 0.005), as well as reduced citrate synthase activity (p = 0.025). Moreover, mutation carriers showed a marked reduction in type 1 muscle fibres (35% vs 62%, p = 0.0005) and increased type 2a fibres (53% vs 32%; p = 0.002) compared with controls. There were no differences in protein content and phosphorylation of 5′ AMP-activated protein kinase, p38 mitogen-activated protein kinase, Erk1 and Erk2.Conclusions/interpretationThese data indicate that inherited insulin resistance coincides with reduced mitochondrial oxidative capacity in a PGC-1α- and muscle fibre type-related manner. Whether this co-existence is directly or indirectly related to insulin resistance remains to be elucidated.

SPARC is up-regulated during skeletal muscle regeneration and inhibits myoblast differentiation

Skeletal muscle repair is mediated primarily by the muscle stem cell, the satellite cell. Several factors, including extracellular matrix, are known to regulate satellite cell function and regeneration. One factor, the matricellular Secreted Protein Acidic and Rich in Cysteine (SPARC) is highly up-regulated during skeletal muscle disease, but its function remains elusive. In the present study, we demonstrate a prominent yet transient increase in SPARC mRNA and protein content during skeletal muscle regeneration that correlates with the expression profile of specific muscle factors like MyoD, Myf5, Myf6, Myogenin, NCAM, CD34, and M-Cadherin, all known to be implicated in satellite cell activation/proliferation following muscle damage. This up regulation was detected in more cell types. Ectopic expression of SPARC in the muscle progenitor cell line C2C12 was performed to mimic the high levels of SPARC seen in muscle disease. SPARC overexpression almost completely abolished myogenic differentiation in these cultures as determined by substantially reduced levels of myogenic factors (Pax7, Myf5, Myod, Mef2B, Myogenin, and Myostatin) and a lack of multinucleated myotubes. These results demonstrate that there is a delicate temporal regulation of SPARC to which more sources in the micro environment contribute, and that disturbances in this, such as extensive up regulation, may have an adverse effect on muscle regeneration.

Effect of testosterone on markers of mitochondrial oxidative phosphorylation and lipid metabolism in muscle of aging men with subnormal bioavailable testosterone.

OBJECTIVE Recent studies have indicated that serum testosterone in aging men is associated with insulin sensitivity and expression of genes involved in oxidative phosphorylation (OxPhos), and that testosterone treatment increases lipid oxidation. Herein, we investigated the effect of testosterone therapy on regulators of mitochondrial biogenesis and markers of OxPhos and lipid metabolism in the skeletal muscle of aging men with subnormal bioavailable testosterone levels. METHODS Skeletal muscle biopsies were obtained before and after treatment with either testosterone gel (n=12) or placebo (n=13) for 6 months. Insulin sensitivity and substrate oxidation were assessed by euglycemic-hyperinsulinemic clamp and indirect calorimetry. Muscle mRNA levels and protein abundance and phosphorylation of enzymes involved in mitochondrial biogenesis, OxPhos, and lipid metabolism were examined by quantitative real-time PCR and western blotting. RESULTS Despite an increase in lipid oxidation (P<0.05), testosterone therapy had no effect on insulin sensitivity or mRNA levels of genes involved in mitochondrial biogenesis (PPARGC1A, PRKAA2, and PRKAG3), OxPhos (NDUFS1, ETFA, SDHA, UQCRC1, and COX5B), or lipid metabolism (ACADVL, CD36, CPT1B, HADH, and PDK4). Consistently, protein abundance of OxPhos subunits encoded by both nuclear (SDHA and UQCRC1) and mitochondrial DNA (ND6) and protein abundance and phosphorylation of AMP-activated protein kinase and p38 MAPK were unaffected by testosterone therapy. CONCLUSION The beneficial effect of testosterone treatment on lipid oxidation is not explained by increased abundance or phosphorylation-dependent activity of enzymes known to regulate mitochondrial biogenesis or markers of OxPhos and lipid metabolism in the skeletal muscle of aging men with subnormal bioavailable testosterone levels.

Aging is associated with diminished muscle re-growth and myogenic precursor cell expansion in the early recovery phase after immobility-induced atrophy in human skeletal muscle

•  Elderly individuals require a prolonged recovery phase in order to return to initial muscle mass levels following short‐term immobilisation. •  The cellular mechanisms responsible for the attenuated re‐growth and associated molecular signalling processes in ageing human skeletal muscle are not fully understood. •  The main study finding was the observation of a less marked muscle mass recovery after immobilisation in elderly compared to young individuals that was paralleled by an elevation in myogenic precursor cell content in young individuals only, whereas the elderly failed to demonstrate any change in myogenic precursor cells. •  No age‐related differences were observed in the expression of major myogenic regulating factors known to promote skeletal muscle hypertrophy or satellite cell proliferation (IGF‐1Ea, MGF, MyoD1, myogenin, HGF gene products). •  In contrast, the expression of myostatin demonstrated a more pronounced up‐regulation following immobilisation along with an attenuated down‐regulation in response to reloading in older compared to young individuals, which may have contributed to the present lack of satellite cell proliferation in ageing muscle.