Muscle-specific microRNAs as biomarkers of Duchenne Muscular Dystrophy progression and response to therapies.

Recent studies have revealed the contribution of fibro-adipogenic progenitors (FAPs) to the pathogenesis and progression of Duchenne Muscular Dystrophy (DMD). While FAPs direct compensatory regeneration at early stages of disease, as the disease progresses they contribute to the progressive replacement of contractile myofibers with fibrotic scars and fatty infiltration. Using the mouse model of DMD - the mdx mice - we have recently reported that FAPs mediate the ability of HDAC inhibitors (HDACi) to promote muscle regeneration and prevent fibro-adipogenic degeneration at early stages of disease. This effect is mediated by the induction of myomiRs that, in turn, target the SWI/SNF components BAF60A and B, thereby favoring the formation of BAF60C-based SWI/SNF complex, which directs the switch from the fibro-adipogenic to the myogenic lineage. Here we show direct evidence of induction of miR-206 and BAF60C, and reduction of BAF60A, in FAPs isolated from mdx muscles exposed to the HDACi Trichostatin A (TSA). We also discuss how increased expression of myomiRs in dystrophic muscles can be integrated with circulating myomiRs to provide accurate biomarkers of disease progression and response to treatment.

Candidate-gene testing for orphan limb-girdle muscular dystrophies.

The term limb-girdle muscular dystrophies (LGMD) identify about two dozens of distinct genetic disorders. Additional genes must play a role, since there are LGMD families excluded from any known locus. The aim of our work is to test a number of candidate genes in unclassified LGMD patient and control DNA samples. We selected the following 11 candidate genes: myozenin 1, 2 and 3, gamma-filamin, kinectin-1, enolase-3 beta, ZASP, TRIM 11 and TRIM 17, OZZ and zeta-sarcoglycan. These candidates were chosen for a combination of different reasons: chromosomal position, sequence homology, interaction properties or muscular dystrophy phenotypes in animal models. The exon and flanking intron sequences were subjected to molecular testing by comparative mutation scanning by HT-DHPLC of LGMD patients versus control. We identified a large number of variations in any of the genes in both patients and controls. Correlations with disease or possible modifying effects on the LGMD phenotype remain to be investigated.

Extensive scanning of the calpain-3 gene broadens the spectrum of LGMD2A phenotypes.

BACKGROUND: The limb girdle muscular dystrophies (LGMD) are a heterogeneous group of Mendelian disorders highlighted by weakness of the pelvic and shoulder girdle muscles. Seventeen autosomal loci have been so far identified and genetic tests are mandatory to distinguish among the forms. Mutations at the calpain 3 locus (CAPN3) cause LGMD type 2A. OBJECTIVE: To obtain unbiased information on the consequences of CAPN3 mutations. PATIENTS: 530 subjects with different grades of symptoms and 300 controls. METHODS: High throughput denaturing HPLC analysis of DNA pools. RESULTS: 141 LGMD2A cases were identified, carrying 82 different CAPN3 mutations (45 novel), along with 18 novel polymorphisms/variants. Females had a more favourable course than males. In 94% of the more severely affected patient group, the defect was also discovered in the second allele. This proves the sensitivity of the approach. CAPN3 mutations were found in 35.1% of classical LGMD phenotypes. Mutations were also found in 18.4% of atypical patients and in 12.6% of subjects with high serum creatine kinase levels. CONCLUSIONS: A non-invasive and cost-effective strategy, based on the high throughput denaturing HPLC analysis of DNA pools, was used to obtain unbiased information on the consequences of CAPN3 mutations in the largest genetic study ever undertaken. This broadens the spectrum of LGMD2A phenotypes and sets the carrier frequency at 1:103.

[An analysis of the variability of the heart rate and its significance in the risk stratification of patients with unstable angina]. / Analisi della variabilità della frequenza cardiaca e suo significato nella stratificazione di rischio dei pazienti con angina instabile.

Decreased heart rate variability (HRV) correlates with increased sympathetic or decreased vagal tone. This could contribute to increase local coronary hyperreactivity caused by atherosclerotic plaque disruption, thus facilitating progression from unstable angina to acute myocardial infarction (AMI). To test this hypothesis we studied 92 patients admitted to the coronary care unit for episodes of chest pain at rest associated with transient ST shifts (> 0.15 mV). Patients who developed AMI in the first 24 hours, as well as those with previous AMI, concomitant valvular or myocardial diseases or diabetes mellitus were not enrolled in the study. Thirty age-matched subjects without any evidence of coronary artery disease were chosen as controls. All patients underwent a 2 to 5 day continuous Holter monitoring during full medical treatment (including beta-blockers, heparin and aspirin). Angiography was performed within 1 week in 88 of the 92 patients. During follow-up (mean duration of 16 +/- 5 days), 26 patients (Group I) had a major coronary event (6 deaths, 7 non fatal AMI, 13 urgent revascularizations). The remaining 66 patients (Group II) had a good clinical outcome. ECG recordings during ST shifts were excluded from Holter monitoring analysis. Time domain measurements of HRV predicted mortality and total events. The most powerful predictors was the standard deviation of the means of the 5 min R-R intervals (SDANN index) which was significantly (p < 0.001) lower in Group I than Group II (55 +/- 18 versus 87 +/- 29).(ABSTRACT TRUNCATED AT 250 WORDS)

[Action of retinol on viable cell recovery and clonogenic potential of HTC cells after in vitro hyperthermia]. / Azione del retinolo sul recupero di cellule vitali e sul potenziale clonogenico di cellule HTC ipertermizzate in vitro.

The aim of this study was to investigate if the association of both hyperthermic and Retinol treatment of HTC hepatoma cells could be useful in antitumor therapy. Treatment with 5 microM Retinol was carried out before or after hyperthermia (42 degrees C or 44 degrees C, one hour; in the latter case it was performed in cells already thermo-selected. We took into consideration two parameters, i.e. the number of the collected vital cells (evaluated by the trypan blue-exclusion test) and the clonal efficiency of these cells (calculated as number of colonies obtained from 250 cells cultured for 5 days). Thermal treatment alone caused a decrease of the number of the collected vital cells and of their clonal efficiency only in the cell cultures incubated at 44 degrees C. Instead the control thermo-selected cells, both at 42 degrees C and at 44 degrees C, showed both decreased clonal efficiency and yield of the vital cells. Compared with the control cultures treated with 0.1% Ethanol, used as vitamin A solvent, only cell cultures treated with Retinol before hyperthermia showed a decreased number of collected viable cells, nevertheless their clonal efficiency was unchanged.

[Effect of hyperthermia and retinol treatment in vitro on HTC cell adhesiveness to laminin and fibronectin]. / Azione dell'ipertermia e del trattamento con retinolo in vitro sull'adesivita' alla laminina ed alla fibronectina delle cellule HTC.

Since cell adhesiveness is very important in the metastatic process and because both hyperthermia and treatment with Retinol can modify the fluidity of the lipid components of the plasma membrane (and therefore its receptor distribution), we investigated if a hyperthermic treatment (at 42 degrees C or 44 degrees C, for one hour) of HTC hepatoma cells, preceded or followed by treatment with 5 microM Retinol, could alter cell adhesiveness to Laminin or to Fibronectin-coated substrata. Hepatoma cells, after such treatments, were collected and processed by Auerbach's method. In the control cells thermal treatment alone caused a decrease of adhesiveness to Laminin but no change in that to Fibronectin. When treatment with Retinol was carried out before hyperthermia, the cells showed an increased adhesiveness to Laminin and a decreased adhesiveness to Fibronectin. Instead, when treatment with Retinol was performed in cells previously thermo-selected, a decrease of adhesiveness to both tested ligands was observed.