Bmi1 enhances skeletal muscle regeneration through MT1-mediated oxidative stress protection in a mouse model of dystrophinopathy

The Polycomb group (PcG) protein Bmi1 is an essential epigenetic regulator of stem cell function during normal development and in adult organ systems. We show that mild up-regulation of Bmi1 expression in the adult stem cells of the skeletal muscle leads to a remarkable improvement of muscle function in a mouse model of Duchenne muscular dystrophy. The molecular mechanism underlying enhanced physiological function of Bmi1 depends on the injury context and it is mediated by metallothionein 1 (MT1)–driven modulation of resistance to oxidative stress in the satellite cell population. These results lay the basis for developing Bmi1 pharmacological activators, which either alone or in combination with MT1 agonists could be a powerful novel therapeutic approach to improve regeneration in muscle wasting conditions.Skeletal muscle is characterized by a remarkable capacity to regenerate after injury, mainly due to the function of satellite cells, the main skeletal muscle stem cells (Brack and Rando, 2012; Wang and Rudnicki, 2012). Polycomb group (PcG) proteins are essential regulators of stem cell function during normal development and in adult organs. They form multi-protein chromatin-associated complexes that play an essential role in the genome-wide epigenetic-mediated remodeling of gene expression during myogenic differentiation of satellite cells, mainly through posttranslational modifications of histones (Asp et al., 2011). Ezh2 and Bmi1 are required for adult satellite cell homeostasis and proliferation in response to muscle injury, an effect mediated at least in part by repression of the ink4a locus (Juan et al., 2011; Robson et al., 2011). Importantly, although Bmi1 is expressed in several types of cancer and its mechanism of action may be similar in a non-neoplastic and neoplastic context, its overexpression does not initiate tumorigenesis (He et al., 2009; Yadirgi et al., 2011).An emerging role for PcG proteins is their involvement in DNA repair (Liu et al., 2009; Facchino et al., 2010; Ismail et al., 2010; Ginjala et al., 2011; Pan et al., 2011). Bmi1^−/−-derived cells show significant mitochondrial dysfunction accompanied by sustained increase in reactive oxygen species (ROS) production that are sufficient to engage the DNA repair pathway (Liu et al., 2009), which is in turn impaired, thus leading to a magnified cellular damage. The balance between intracellular ROS and antioxidant molecules is vital in determining the rate of oxidative damage accumulation and the impaired function of satellite cells in aging and in myopathies, in which decreased anti-oxidative capacity has been documented (Fulle et al., 2005; Whitehead et al., 2006; Tidball and Wehling-Henricks, 2007). X-linked Duchenne muscular dystrophy (DMD) is the most common primary myopathy caused by the loss of the dystrophin protein from the plasma membrane, which causes loss of its integrity and fiber damage during repeated cycles of muscle degeneration and regeneration (Duncan, 1989). The proliferative capacity of myogenic cells was reported to be rapidly exhausted in dystrophin-deficient muscle, also because they are more sensitive to oxidative stress injury, leading to reduced and defective regeneration of the muscle as the disease progresses (Blau et al., 1983, 1985; Disatnik et al., 1998). Moreover, enzymatic adaptations to exercise-induced production of ROS and free radical damage are significantly decreased in dystrophic compared with normal muscles (Faist et al., 1998, 2001). Overall, an impaired protection against ROS in dystrophic muscle appears to contribute to disease progression as also indicated by the beneficial, albeit transient, effect of antioxidants in ameliorating the skeletal muscle pathophysiology of DMD patients (Whitehead et al., 2008).Metallothionein 1 (MT1) and MT2 are ubiquitously expressed (Kägi and Hunziker, 1989) low molecular weight, cysteine–rich zinc binding proteins. Although the role of MT1 in promoting cell proliferation is controversial (Smith et al., 2008), studies on MT-null liver cells showed their failure to regenerate after oxidative stress injury (Oliver et al., 2006).Here, we show that overexpression of Bmi1 in the satellite cells significantly improves muscle strength through enhanced MT1-mediated protection of these cells from oxidative stress in a mouse model of dystrophinopathies but not after acute traumatic injury.     

Cardiac magnetic resonance features of left dominant arrhythmogenic cardiomyopathy: differential diagnosis with myocarditis

The International Journal of Cardiovascular Imaging - Cardiac magnetic resonance (CMR) findings suggesting a suspected left-dominant arrhythmogenic cardiomyopathy (LDAC) may be difficult to...     

Acute administration of alprazolam, a benzodiazepine activating GABA receptors, inhibits cortisol secretion in patients with subclinical but not overt Cushing’s syndrome

The purpose of this study is to verify whether acute pre-treatment with alprazolam (ALP), a benzodiazepine that inhibits HPA secretion in normal subjects, could better characterize patients with subclinical Cushing’s syndrome (SCS) than the 1-mg dexamethasone test (DST). In 22 patients with SCS, 10 with overt Cushing’s syndrome (CS), 11 with non-functioning adrenal incidentalomas (NF) and 14 normal subjects (NS) we studied the effect of ALP (1 mg, p.o. at 2300 hours) on cortisol levels after 1-mg DST. Cortisol levels (mean ± SEM) after DST were lower (P = 0.012) in SCS (3.9 ± 0.3 μg/dl) than in overt CS (10.4 ± 1.9 μg/dl), while they were higher (P = 0.0005) than in NF (1.1 ± 0.1 μg/dl) and NS (1.5 ± 0.1 μg/dl). After ALP pre-treatment, cortisol levels further decreased (P = 0.004) in SCS (3.0 ± 0.3 μg/dl), but neither in CS (9.3 ± 1.3 μg/dl) nor in NF (1.3 ± 0.1 μg/dl) and in NS (1.3 ± 0.1 μg/dl). In SCS, cortisol levels after ALP + 1-mg DST persisted lower (P = 0.0005) than those in CS, but higher (P = 0.0005) than those in NF and NS. Considering individual cases, ALP pre-treatment reduced cortisol levels <3 and <1.8 μg/dl in 50 and 23 % of SCS patients, respectively. ALP amplifies the cortisol inhibition exerted by 1-mg DST in patients with SCS but not in those with CS. The clinical usefulness of ALP to increase the sensitivity of 1-mg DST to identify true autonomous cortisol release in patients with adrenal incidentalomas as well as to predict different clinical outcomes remains to be clarified.     

Clinical characteristics, timing of peak responses and safety aspects of two dosing regimens of the glucagon stimulation test in evaluating growth hormone and cortisol secretion in adults

Weight-based (WB: 0.03 mg/kg) and fixed dose (FD: 1–1.5 mg) regimens of the glucagon stimulation test (GST) have been used to evaluate GH and cortisol secretion in children and adults, respectively. However, experience of the WB regimen in assessing GH and cortisol secretion in adults are limited. We describe a multicenter experience using WB and FD regimens in evaluating GH and cortisol secretion in adults suspected of GH deficiency and central adrenal insufficiency. Retrospective case series of GSTs (n = 515) performed at five tertiary centers. Peak and nadir glucose, and peak GH and peak cortisol responses occurred later with WB (mean dose: 2.77 mg) compared to FD (mean dose: 1.20 mg) regimens. Main side-effects were nausea and vomiting, particularly in younger females. Nausea was comparable but vomiting was more frequent in the WB regimen (WB: 10.0 % vs FD: 2.4 %; P < 0.05). Peak and nadir glucose, ΔGH, and peak and Δcortisol were higher in the WB regimen. In both regimens, age correlated negatively with peak cortisol levels, and body mass index (BMI), fasting, peak and nadir glucose correlated negatively with peak GH levels. WB and FD regimens can induce adult GH and cortisol secretion, but peak responses occur later in the WB regimen. Both regimens are relatively safe, and vomiting was more prevalent in the WB regimen. As age, BMI, and glucose tolerance negatively correlated with peak GH and cortisol levels, the WB regimen may be more effective than the FD regimen in older overweight glucose intolerant patients.     

Revisiting hyper- and hypo-androgenism by tandem mass spectrometry

Modern endocrinology is living a critical age of transition as far as laboratory testing and biochemical diagnosis are concerned. Novel liquid chromatography—tandem mass spectrometry (LC-MS/MS) assays for steroid measurement in biological fluids have abundantly demonstrated their analytical superiority over immunometric platforms that until now have dominated the world of steroid hormones determination in clinical laboratories. One of the most useful applications of LC-MS/MS is in the hypogonadism and hyperandrogenism field: LC-MS/MS has proved particularly suitable for the detection of low levels of testosterone typical of women and children, and in general more reliable in accurately determining hypogonadal male levels. This technique also offers increased informative power by allowing multi-analytical profiles that give a more comprehensive picture of the overall hormonal asset. Several LC-MS/MS methods for testosterone have been published in the last decade, some of them included other androgen or more comprehensive steroid profiles. LC-MS/MS offers the concrete possibility of achieving a definitive standardization of testosterone measurements and the generation of widely accepted reference intervals, that will set the basis for a consensus on the diagnostic value of biochemical testing. The present review is aimed at summarizing technological advancements in androgen measurements in serum and saliva. We also provide a picture of the state of advancement of standardization of testosterone assays, of the redefinition of androgen reference intervals by novel assays and of studies using LC-MS/MS for the characterization and diagnosis of female hyperandrogenism and male hypogonadism.