Aging in human liver and skeletal muscle: studies on proteasomes

Aging is a complex phenomenon that affects organs and tissues at a different rate. With advancing age, the skeletal muscle undergoes a progressive loss of mass and strength, a process known as sarcopenia that leads to a decreased mobility and increased risk of falls and invalidity. On the other side, another organ such as the liver that is endowed with a peculiar regenerative capacity seems to be only marginally affected by aging. Accordingly, clinical data indicate that liver transplantation from aged subjects has, in specific conditions, function and duration comparable to those achievable with grafts of liver from young donors. The molecular mechanisms involved in these peculiar aging patterns are still largely unknown, but it is conceivable that protein degradation machineries might play an important role, as they are responsible for the maintenance of cellular homeostasis. Indeed, it has been suggested that alteration of proteostasis may contribute to the onset and progression of several age-related pathological conditions, including skeletal muscle wasting and sarcopenia, as well as to the aging phenotypes. The ubiquitin-proteasome system (UPS) is one of the most important cellular pathways for intracellular degradation of short-lived as well as damaged proteins. To date, studies on the age-related modifications of proteasomes in liver and skeletal muscle were performed prevalently in rodents, with controversial results, while only preliminary observations have been obtained in human liver and skeletal muscle. In this scenario, we want to investigate and characterize in humans the age-related modifications of proteasomes of these two different organs.

The Influence of Baseline Metabolic Rate and Exercise Training on Cardio-Respiratory Dynamics

Speeding the VO2 kinetics results in a reduction of the O2 deficit. Two factors might determine VO2 kinetics: oxygen delivery to muscle (Tschakovsky and Hughson 1999) and a muscle 'metabolic inertia' (Grassi et al. 1996). Therefore, in study 1 we investigated VO2 kinetics and cardiovascular system adaptations during step exercise transitions in different regions of the moderate domain. In study 2 we investigated muscle oxygenation and cardio-pulmonary adaptations during step exercise tests before, after and over a period of training. Study 1 methods: Seven subjects (26 ± 8 yr; 176 ± 5 cm; 69 ± 6 kg) performed 4 types of step transition from rest (0-50W; 0-100W) or elevate baseline (25-75W; 25-125W). GET and VO2max were assessed before testing. O2 uptake and were measured during testing. Study 2 methods: 10 subjects (25 ± 4 yr; 175 ± 9 cm; 71 ± 12 kg) performed a step transition test (0 to 100 W) before, after and during 4 weeks of endurance training (ET). VO2max and GET were assessed before and after of ET (40 minutes, 3 times a week, 60% O2max). VO2 uptake, Q and deoxyheamoglobin were measured during testing. Study 1 results: VO2 τ and the functional gain were slower in the upper regions of the moderate domain. Q increased more abruptly during rest to work condition. Q τ was faster than VO2 τ for each exercise step. Study 2 results: VO2 τ became faster after ET (25%) and particularly after 1 training session (4%). Q kinetics changed after 4 training sessions nevertheless it was always faster than VO2 τ. An attenuation in ∆[HHb] /∆VO2 was detectible. Conclusion: these investigations suggest that muscle fibres recruitment exerts a influence on the VO2 response within the moderate domain either during different forms of step transition or following ET.

Bioengineering of exercise: biomechanical and metabolic aspects

The field of research of this dissertation concerns the bioengineering of exercise, in particular the relationship between biomechanical and metabolic knowledge. This relationship can allow to evaluate exercise in many different circumstances: optimizing athlete performance, understanding and helping compensation in prosthetic patients and prescribing exercise with high caloric consumption and minimal joint loading to obese subjects. Furthermore, it can have technical application in fitness and rehabilitation machine design, predicting energy consumption and joint loads for the subjects who will use the machine. The aim of this dissertation was to further understand how mechanical work and metabolic energy cost are related during movement using interpretative models. Musculoskeletal models, when including muscle energy expenditure description, can be useful to address this issue, allowing to evaluate human movement in terms of both mechanical and metabolic energy expenditure. A whole body muscle-skeletal model that could describe both biomechanical and metabolic aspects during movement was identified in literature and then was applied and validated using an EMG-driven approach. The advantage of using EMG driven approach was to avoid the use of arbitrary defined optimization functions to solve the indeterminate problem of muscle activations. A sensitivity analysis was conducted in order to know how much changes in model parameters could affect model outputs: the results showed that changing parameters in between physiological ranges did not influence model outputs largely. In order to evaluate its predicting capacity, the musculoskeletal model was applied to experimental data: first the model was applied in a simple exercise (unilateral leg press exercise) and then in a more complete exercise (elliptical exercise). In these studies, energy consumption predicted by the model resulted to be close to energy consumption estimated by indirect calorimetry for different intensity levels at low frequencies of movement. The use of muscle skeletal models for predicting energy consumption resulted to be promising and the use of EMG driven approach permitted to avoid the introduction of optimization functions. Even though many aspects of this approach have still to be investigated and these results are preliminary, the conclusions of this dissertation suggest that musculoskeletal modelling can be a useful tool for addressing issues about efficiency of movement in healthy and pathologic subjects.

Different approaches to identify markers for meat quality and production traits in pigs: analysis of the transcriptome in post mortem skeletal muscle and investigation of candidate genes

Due to the growing attention of consumers towards their food, improvement of quality of animal products has become one of the main focus of research. To this aim, the application of modern molecular genetics approaches has been proved extremely useful and effective. This innovative drive includes all livestock species productions, including pork. The Italian pig breeding industry is unique because needs heavy pigs slaughtered at about 160 kg for the production of high quality processed products. For this reason, it requires precise meat quality and carcass characteristics. Two aspects have been considered in this thesis: the application of the transcriptome analysis in post mortem pig muscles as a possible method to evaluate meat quality parameters related to the pre mortem status of the animals, including health, nutrition, welfare, and with potential applications for product traceability (chapters 3 and 4); the study of candidate genes for obesity related traits in order to identify markers associated with fatness in pigs that could be applied to improve carcass quality (chapters 5, 6, and 7). Chapter three addresses the first issue from a methodological point of view. When we considered this issue, it was not obvious that post mortem skeletal muscle could be useful for transcriptomic analysis. Therefore we demonstrated that the quality of RNA extracted from skeletal muscle of pigs sampled at different post mortem intervals (20 minutes, 2 hours, 6 hours, and 24 hours) is good for downstream applications. Degradation occurred starting from 48 h post mortem even if at this time it is still possible to use some RNA products. In the fourth chapter, in order to demonstrate the potential use of RNA obtained up to 24 hours post mortem, we present the results of RNA analysis with the Affymetrix microarray platform that made it possible to assess the level of expression of more of 24000 mRNAs. We did not identify any significant differences between the different post mortem times suggesting that this technique could be applied to retrieve information coming from the transcriptome of skeletal muscle samples not collected just after slaughtering. This study represents the first contribution of this kind applied to pork. In the fifth chapter, we investigated as candidate for fat deposition the TBC1D1 [TBC1 (tre-2/USP6, BUB2, cdc16) gene. This gene is involved in mechanisms regulating energy homeostasis in skeletal muscle and is associated with predisposition to obesity in humans. By resequencing a fragment of the TBC1D1 gene we identified three synonymous mutations localized in exon 2 (g.40A>G, g.151C>T, and g.172T>C) and 2 polymorphisms localized in intron 2 (g.219G>A and g.252G>A). One of these polymorphisms (g.219G>A) was genotyped by high resolution melting (HRM) analysis and PCR-RFLP. Moreover, this gene sequence was mapped by radiation hybrid analysis on porcine chromosome 8. The association study was conducted in 756 performance tested pigs of Italian Large White and Italian Duroc breeds. Significant results were obtained for lean meat content, back fat thickness, visible intermuscular fat and ham weight. In chapter six, a second candidate gene (tribbles homolog 3, TRIB3) is analyzed in a study of association with carcass and meat quality traits. The TRIB3 gene is involved in energy metabolism of skeletal muscle and plays a role as suppressor of adipocyte differentiation. We identified two polymorphisms in the first coding exon of the porcine TRIB3 gene, one is a synonymous SNP (c.132T> C), a second is a missense mutation (c.146C> T, p.P49L). The two polymorphisms appear to be in complete linkage disequilibrium between and within breeds. The in silico analysis of the p.P49L substitution suggests that it might have a functional effect. The association study in about 650 pigs indicates that this marker is associated with back fat thickness in Italian Large White and Italian Duroc breeds in two different experimental designs. This polymorphisms is also associated with lactate content of muscle semimembranosus in Italian Large White pigs. Expression analysis indicated that this gene is transcribed in skeletal muscle and adipose tissue as well as in other tissues. In the seventh chapter, we reported the genotyping results for of 677 SNPs in extreme divergent groups of pigs chosen according to the extreme estimated breeding values for back fat thickness. SNPs were identified by resequencing, literature mining and in silico database mining. analysis, data reported in the literature of 60 candidates genes for obesity. Genotyping was carried out using the GoldenGate (Illumina) platform. Of the analyzed SNPs more that 300 were polymorphic in the genotyped population and had minor allele frequency (MAF) >0.05. Of these SNPs, 65 were associated (P<0.10) with back fat thickness. One of the most significant gene marker was the same TBC1D1 SNPs reported in chapter 5, confirming the role of this gene in fat deposition in pig. These results could be important to better define the pig as a model for human obesity other than for marker assisted selection to improve carcass characteristics.

The regulation of Satellite Cells during skeletal muscle regeneration and neuromuscular disease.

Skeletal muscle possesses the remarkable capacity to complete a rapid and extensive regeneration, even following severe damage. The regenerative ability of skeletal muscle relies on Satellite Cells (SCs), a population of muscle specific adult stem cells. However, during aging or under several pathological conditions, the ability of skeletal muscle to fully regenerated is compromised. Here, a morphological and molecular study on SCs from patients affected by ALS is described. Moreover, the role of the cell cycle regulator P16Ink4a during skeletal muscle regeneration and aging has been investigated.