Vito Pesce

Age-related mitochondrial genotypic and phenotypic alterations in human skeletal muscle.

To have a clearer picture of how mitochondrial damages are associated to aging, a comprehensive study of phenotypic and genotypic alterations was carried out, analyzing with histochemical and molecular biology techniques the same skeletal muscle specimens of a large number of healthy subjects from 13 to 92 years old. Histochemical data showed that ragged red fibers (RRF) appear at about 40 years of age and are mostly cytochrome c oxidase (COX)-positive, whereas they are almost all COX-negative thereafter. Molecular analyses showed that the 4977 bp deletion of mitochondrial DNA (mtDNA(4977)) and the 7436 bp deletion of mtDNA (mtDNA(7436)) are already present in individuals younger than 40 years of age, but their occurrence does not change with age. After 40 years of age the number of mtDNA deleted species, as revealed by Long Extension PCR (LX-PCR), increases, the 10422 bp deletion of mtDNA (mtDNA(10422)) appears, although with a very low frequency of occurrence, and mtDNA content is more than doubled. Furthermore, mtDNA(4977) level directly correlates with that of COX-negative fibers in the same analyzed subjects. These data clearly show that, after 40 years of age, the phenotypic and genotypic mitochondrial alterations here studied appear in human skeletal muscle and that they are closely related.

Increased expression of mitochondrial transcription factor A and nuclear respiratory factor-1 in skeletal muscle from aged human subjects.

The expression of two factors involved in the nuclear–mitochondrial crosstalk, namely the mitochondrial transcription factor A (TFAM) and the nuclear respiratory factor‐1 (NRF‐1), was studied in human skeletal muscle biopsies of young and aged subjects. Aged subjects presented a 2.6‐fold and an 11‐fold increase of the levels of TFAM protein and TFAM mRNA, respectively. The increased expression of TFAM was associated to the doubling of NRF‐1 DNA‐binding affinity and to a 6‐fold increase of NRF‐1 mRNA level. The upregulation of TFAM and NRF‐1, in aged skeletal muscle, appears involved in the pathway leading to the age‐related increase of mitochondrial DNA content.

Accumulation of overoxidized Peroxiredoxin III in aged rat liver mitochondria.

Overoxidation and subsequent inactivation of Peroxiredoxin III (PrxIII), a mitochondrial H(2)O(2) scavenging enzyme, have been reported in oxidative stress conditions. No data are available in the literature about the presence of overoxidized forms of PrxIII in aged tissues. Liver mitochondria from 12-month-old rats and 28-month-old rats were here analyzed by two-dimensional gel electrophoresis. A spot corresponding to the native form of PrxIII was present in adult and old rats with the same volume, whereas an additional, more acidic spot, of the same molecular weight of the native form, accumulated only in old rats. The acidic spot was identified, by MALDI-MS analysis, as a form of PrxIII bearing the cysteine of the catalytic site overoxidized to sulphonic acid. This modified PrxIII form corresponds to the irreversibly inactivated enzyme, here reported, for the first time, in aging. Three groups of 28-month-old rats treated with acetyl-l-carnitine were also examined. Reduced accumulation of the overoxidized PrxIII form was found in all ALCAR-treated groups.

A DIGE approach for the assessment of rat soleus muscle changes during unloading: effect of acetyl‐L‐carnitine supplementation

After hind limb suspension, a remodeling of postural muscle phenotype is observed. This remodeling results in a shift of muscle profile from slow‐oxidative to fast‐glycolytic. These metabolic changes and fiber type shift increase muscle fatigability. Acetyl‐L‐carnitine (ALCAR) influences the skeletal muscle phenotype of soleus muscle suggesting a positive role of dietary supplementation of ALCAR during unloading. In the present study, we applied a 2‐D DIGE, mass spectrometry and biochemical assays, to assess qualitative and quantitative differences in the proteome of rat slow‐twitch soleus muscle subjected to disuse. Meanwhile, the effects of ALCAR administration on muscle proteomic profile in both unloading and normal‐loading conditions were evaluated. The results indicate a modulation of troponin I and tropomyosin complex to regulate fiber type transition. Associated, or induced, metabolic changes with an increment of glycolytic enzymes and a decreased capacity of fat oxidation are observed. These metabolic changes appear to be counteracted by ALCAR treatment, which restores the mitochondrial mass and decreases the glycolytic enzyme expression, suggesting a normalization of the metabolic shift observed in unloaded animals. This normalization is accompanied by a maintenance of body weight and seems to prevent a switch of fiber type.

The PGC-1α-dependent pathway of mitochondrial biogenesis is upregulated in type I endometrial cancer

PGC-1alpha-dependent pathway of mitochondrial biogenesis was investigated for the first time in type I endometrial cancer and in normal endometrium. In cancer endometrial tissue the citrate synthase activity, the mitochondrial DNA content and the TFAM level were found doubled compared to control endometrial tissue. Moreover, a 1.6- and 1.8-fold increase, respectively, of NRF-1 and PGG-1alpha expression was found. This study demonstrates, for the first time, that the increased mitochondrial biogenesis in type I endometrial cancer is associated to the upregulation of PGC-1alpha signalling pathway.

Aging and calorie restriction oppositely affect mitochondrial biogenesis through TFAM binding at both origins of mitochondrial DNA replication in rat liver.

Aging affects mitochondria in a tissue-specific manner. Calorie restriction (CR) is, so far, the only intervention able to delay or prevent the onset of several age-related changes also in mitochondria. Using livers from middle age (18-month-old), 28-month-old and 32-month-old ad libitum-fed and 28-month-old calorie-restricted rats we found an age-related decrease in mitochondrial DNA (mtDNA) content and mitochondrial transcription factor A (TFAM) amount, fully prevented by CR. We revealed also an age-related decrease, completely prevented by CR, for the proteins PGC-1α NRF-1 and cytochrome c oxidase subunit IV, supporting the efficiency of CR to forestall the age-related decrease in mitochondrial biogenesis. Furthermore, CR counteracted the age-related increase in oxidative damage to proteins, represented by the increased amount of oxidized peroxiredoxins (PRX-SO3) in the ad libitum-fed animals. An unexpected age-related decrease in the mitochondrial proteins peroxiredoxin III (Prx III) and superoxide dismutase 2 (SOD2), usually induced by increased ROS and involved in mitochondrial biogenesis, suggested a prevailing relevance of the age-reduced mitochondrial biogenesis above the induction by ROS in the regulation of expression of these genes with aging. The partial prevention of the decrease in Prx III and SOD2 proteins by CR also supported the preservation of mitochondrial biogenesis in the anti-aging action of CR. To investigate further the age- and CR-related effects on mitochondrial biogenesis we analyzed the in vivo binding of TFAM to specific mtDNA regions and demonstrated a marked increase in the TFAM-bound amounts of mtDNA at both origins of replication with aging, fully prevented by CR. A novel, positive correlation between the paired amounts of TFAM-bound mtDNA at these sub-regions was found in the joined middle age ad libitum-fed and 28-month-old calorie-restricted groups, but not in the 28-month-old ad libitum-fed counterpart suggesting a quite different modulation of TFAM binding at both origins of replication in aging and CR.

Hip Fracture in a Patient Affected by Transient Osteoporosis of the Femoral Head During the Last Trimester of Pregnancy

Few cases of hip fracture in pregnant women affected by transient osteoporosis of the femoral head have been reported in the literature, but its real incidence seems to be underestimated. During pregnancy, osteoporosis manifests itself with an insidious onset of hip pain and limp without any trauma or infective episode in clinical history. Its clinical course is characterized by spontaneous recovery a few weeks to several months after delivery. This article describes the case of a 35-year-old woman with a sudden onset of bilateral hip pain during the last trimester of her first pregnancy; she had neither history of steroid therapy nor alcohol abuse; her body temperature and serological parameters were normal. Bilateral transient osteoporosis of the femoral heads was suspected and confirmed by magnetic resonance imaging. Fifteen days postpartum, she was admitted to our clinic with a displaced femoral neck fracture. A cementless total hip arthroplasty was performed to quickly begin a rehabilitative program. She underwent antiresorptive therapy with alendronic acid 70 mg/week and vitamin D for 3 months. Three months after the fracture, a dual-energy x-ray absorptiometry scan showed osteopenia (T-score, -1.5). Risedronic acid 35 mg/week and vitamin D were then prescribed. The last physical examination at 3 months postoperatively revealed a gradual recovery of the autonomy in activities of daily life.

Comparative efficacy of different doses of fibrin sealant to reduce bleeding after total knee arthroplasty.

In recent years, the application of fibrin sealant has been shown to be efficacious in the management of intraoperative and postoperative hemostasis. We designed a prospective randomized controlled clinical trial to assess the efficacy of two different dosages of fibrin sealant (5 and 10 ml) in patients undergoing total knee arthroplasty, as compared with the untreated control group. A total of 90 patients entered the study, randomly assigned to one of the three groups: treatment with 5 ml fibrin sealant (30 patients), with 10 ml fibrin sealant (30 patients) or no treatment (30 patients). A statistically significant difference in the mean reduction of hemoglobin concentrations was found on the first postoperative day in the treated groups as compared with the controls: 5 ml fibrin sealant (2.6 mg/dl) and 10 ml fibrin sealant (2.5 mg/dl) vs. controls (3.7 mg/dl) (P = 0.024). The mean number of blood transfusions was significantly lower in the treated groups: 5 ml (0.5) and 10 ml (0.3) of fibrin sealant vs. controls (1) (P = 0.0019). Functional recovery was also better in the treated groups: on the seventh day, the joint ROM (range of motion) was statistically superior in the 5 ml group (96.5°) and 10 ml group (98.8°) as compared with the controls (75.5°) (P < 0.0001). This study supports the efficacy of administering fibrin sealant in the perioperative management of bleeding after total knee arthroplasty and shows that a 5 ml dosage yields a comparable outcome to the 10 ml dosage previously reported in the literature.

Mitochondrial DNA content and mass increase in progression from normal to hyperplastic to cancer endometrium

BackgroundAn increase in mitochondrial DNA (mtDNA) content and mitochondrial biogenesis associated with the activation of PGC-1α signalling pathway was previously reported in type I endometrial cancer. The aim of this study has been to evaluate if mtDNA content and the citrate synthase (CS) activity, an enzyme marker of mitochondrial mass, increase in progression from control endometrium to hyperplasia to type I endometrial carcinoma.ResultsGiven that no statistically significant change in mtDNA content and CS activity in endometrium taken from different phases of the menstrual cycle or in menopause was found, these samples were used as control. Our research shows, for the first time, that mtDNA content and citrate synthase activity increase in hyperplastic endometrium compared to control tissues, even if their levels remain lower compared to cancer tissue. In particular, mtDNA content increases seem to precede increases in CS activity. No statistically significant change in mtDNA content and in CS activity was found in relation to different histopathological conditions such as grade, myometrial invasion and stage.ConclusionMtDNA content and citrate synthase activity increases in pre-malignant lesions could be a potential molecular marker for progression from hyperplasia to carcinoma.

I-ONE therapy in patients undergoing total knee arthroplasty: a prospective, randomized and controlled study

BackgroundTotal knee arthroplasty (TKA) is often associated with a severe local inflammatory reaction which, unless controlled, leads to persistent pain up to one year after surgery. Standard and accelerated rehabilitation protocols are currently being implemented after TKA, but no consensus exists regarding the long-term effects. Biophysical stimulation with pulsed electromagnetic fields (PEMFs) has been demonstrated to exert an anti-inflammatory effect, to promote early functional recovery and to maintain a positive long-term effect in patients undergoing joint arthroscopy. The aim of this study was to evaluate whether PEMFs can be used to limit the pain and enhance patient recovery after TKA.MethodsA prospective, randomized, controlled study in 30 patients undergoing TKA was conducted. Patients were randomized into experimental PEMFs or a control group. Patients in the experimental group were instructed to use I-ONE stimulator 4hours/day for 60days. Postoperatively, all patients received the same rehabilitation program. Treatment outcome was assessed using the Knee Society Score, SF-36 Health-Survey and VAS. Patients were evaluated pre-operatively and one, two, six and 12 months after TKA. Joint swelling and Non Steroidal Anti Inflammatory Drug (NSAID) consumption were recorded. Comparisons between the two groups were carried out using a two-tail heteroschedastic Student’s t-test. Analysis of variance for each individual subject during the study was performed using ANOVA for multiple comparisons, applied on each group, and a Dunnet post hoc test. A p value < 0.05 was considered statistically significant.ResultsPre-operatively, no differences were observed between groups in terms of age, sex, weight, height, Knee-Score, VAS, SF-36 and joint swelling, with the exception of the Functional Score. The Knee-Score, SF-36 and VAS demonstrated significantly positive outcomes in the I-ONE stimulated group compared with the controls at follow-ups. In the I-ONE group, NSAID use was reduced and joint swelling resolution was more rapid than in controls. The effect of I-ONE therapy was maintained after use of the device was discontinued.ConclusionsThe results of the study show early functional recovery in the I-ONE group. I-ONE therapy should be considered after TKA to prevent the inflammatory reaction elicited by surgery, for pain relief and to speed functional recovery.Trial registrationCurrent Controlled Trials ISRCTN10526056