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The ubiquitin proteasome pathway is the predominant biological mechanism of myofibrillar protein (MF) degradation.  To test the hypothesis that amino acid and insulin act synergistically to regulate proteolysis, two experimental models were employed;  an <i>in vivo </i>study on growing calves and an <i>in vitro</i> C2C12 myotubes culture. Calves growing at 0.3kg/day, were constantly infused with glucose at a low (LDG) or high (HDG) dose (to stimulate insulin) with or without essential amino acids (EAA).  Glucose infusions increased plasma insulin and IGF-1 concentrations in a dose dependent manner (P<0.05).  HDG was associated with decreased plasma urea nitrogen and 3-MH concentrations and 3-MH:creatinine output (an index of MF degradation) (P < 0.05).  Glucose infusions down regulated the expression of 14-kDa E2 ubiquitin conjugating enzyme and C2 20 S proteasome sub unit, however EAA did not alter the effect of raised plasma insulin on muscle ubiquitin proteasome pathway suggesting that under the conditions employed, EAA do not act synergistically with insulin to decrease myofibrillar protein degradation, <i>in vivo.</i> In the <i>in vitro</i> experiments, amino acid deprivation (0.2 X physiological concentration amino acid;  PC AA) of myotubes for 8 h was associated with increased (P < 0.05) proteolysis (measured from TCA soluble ³H-tyrosine release in the medium), compared to controls (1.0 X PC AA).  Addition of insulin inhibited this increase (P < 0.05).  Rapamycin significantly increased proteolysis in 1.0 X PC AA media suggesting amino acid might regulate proteolysis through mTOR signalling pathway.  Reduced amino acid supply also increased 14-kDa E2 and C2 mRNA expression compared to controls (P < 0.05).  Increasing leucine concentration in 0.2 X PC AA basal media showed a dose dependent decrease in protein degradation and expression of 14-kDa E2, in the presence of insulin.  In conclusion, the results suggested that decreased availability of amino acids was associated with increased total proteolysis and that anti-catabolic effect of amino acid in C2C12 muscle cell cultures, was additive to that of insulin.

Myotilin is a muscle-specific Z-disc protein with putative roles in myofibril assembly and structural upkeep of the sarcomere. Several myotilin point mutations have been described in patients with Limb-Girdle Muscular Dystrophy Type 1A (LGMD1A), myofibrillar myopathy (MFM), spheroid body myopathy (SBM), and distal myopathy, four similar adult-onset, progressive, and autosomal dominant muscular dystrophies--collectively called the myotilinopathies. It is not yet known how myotilin mutations cause muscle disease. To investigate myotilin's role in the pathogenesis of muscle disease, I have created and characterized transgenic mice expressing mutant (Thr57Ile) myotilin under the control of the human skeletal alpha-actin promoter. Like LGMD1A and MFM patients, these mice develop progressive myofibrillar pathology that includes Z-disc streaming, excess myofibrillar vacuolization, and plaque-like myofibrillar aggregation. These aggregates become progressively larger and more numerous with age. I show that the mutant myotilin protein properly localizes to the Z-disc, and also heavily populates the aggregates, along with several other Z-disc associated proteins. Whole muscle physiological analysis reveals that the extensor digitorum longus (EDL) muscle of transgenic mice exhibits significantly reduced maximum specific isometric force compared to littermate controls. Intriguingly, the soleus and diaphragm muscles are spared of any abnormal myopathology and show no reductions in maximum specific force. These data provide evidence that myotilin mutations promote aggregate-dependent contractile dysfunction. To better understand myotilin function, I also created two separate lines of myotilin domain deletion transgenic mice: one expresses a deletion of the N-terminal domain and the second expresses a deletion of the minimal alpha-actinin binding site. Studies in these mice show that 1) the N-terminal domain of myotilin may be required for normal localization to the Z-disc; 2) interaction with alpha-actinin is not required for localization of myotilin to the Z-disc; and 3) deletion of the alpha-actinin binding site causes an aggregation phenotype similar to that of the TgT57I mouse and myotilinopathy patients. In sum, I have established a promising patho-physiological mouse model that unifies the diverse clinical phenotypes of the myotilinopathies. This mouse model promises to be a key resource for understanding myotilin function, unraveling LGMD1A pathogenesis, and investigating therapeutics. / Dissertation

Both resistance and endurance exercise elicit an increase in muscle protein synthesis during recovery from exercise. Ingestion of amino acids augments the exercise-induced stimulation of muscle protein synthesis following resistance exercise. Our work showed that 8 wk of unilateral resistance training induced muscle hypertrophy only in the exercised limb. Importantly, using this unilateral model we showed that muscle hypertrophy was confined to the exercised leg and occurred without measurable changes in circulating anabolic hormones. We then went on to use the unilateral leg resistance exercise model to study how animal-derived (milk) and plant-derived (soy) proteins impacted acute post-exercise protein turnover. We observed that ingestion of soy or milk protein resulted in a positive net protein balance following resistance exercise. Moreover, milk promoted a greater net protein balance and muscle protein synthesis than soy protein. In the final study, a key finding was that acute endurance and resistance exercise differentially stimulated myofibrillar and mitochondrial protein synthesis and also differentially affected cellular signaling proteins involved in the regulation of the protein synthetic response. Specifically, the acute, untrained state response showed that resistance exercise stimulated myofibrillar and mitochondrial protein synthesis while endurance exercise stimulated mitochondrial protein synthesis. Following resistance training only myofibrillar protein synthesis increased after exercise, while mitochondrial protein synthesis was unchanged. Endurance exercise training did not affect the acute protein synthetic response and so following training mitochondrial protein synthesis was stimulated as it was acutely, prior to training. In conclusion, the studies within this thesis provided novel insights on the impact of intact dietary proteins and differing modes of exercise on the control skeletal muscle protein metabolism. / Thesis / Doctor of Philosophy (PhD)

Orientador: Luis Arthur Loyola Chardulo / Banca: Antonio Carlos Silveira / Banca: Albino Luchiari Filho / Resumo: O experimento foi realizado no Setor de Confinamento de Gado de Corte da Faculdade de Medicina Veterinária e Zootecnia e no Laboratório de Bioquímica de Proteínas do Instituto de Biociências. Foram utilizados animais da raça Nelore, mestiços u Aberdeen X Nelore e mestiços u Simental X Nelore, abatidos com idade entre 12 e 15 meses conforme estabelecido pelo modelo biológico superprecoce. O experimento foi conduzido em um delineamento inteiramente casualizado. O objetivo do presente estudo foi análise de componentes da maciez de novilhos superprecoces de grupos genéticos distintos. Não foi verificada diferença estatística (p>0,01) entre os grupos genéticos para a força de cisalhamento, Índice de Fragmentação Miofibrilar (MFI) e frações do colágeno, entretanto houve influência (p<0,01) do período postmortem, exceto para o colágeno. A carne de animais abatidos entre 12 e 15 meses de idade apresenta atributos de qualidade independente do grupo genético utilizado e com sete dias de maturação todos os animais apresentaram carne com grau de maciez desejável. / Abstract: The experiment was accomplished in the Section of Feedlot of cattle of Faculdade de Medicina Veterinária e Zootecnia and in the Laboratório de Bioquímica de Proteínas do Instituito de Biociências. Nelore breed, u Aberdeen X Nelore crossbreed and u Simental X Nelore crossbreed were used and slaughtered accordingly with the brazilian system called "superprecoce". The experiment was accomplished in a completely randomized design. The objective of the present study was the evaluation of tenderness components of "superprecoce" of different genetic groups. There was no statistics difference (p>0,01) between genetic groups for the shear force values, Myofibrillar Fragmentation Index (MFI) and collagen, however there was influence (p<0,01) of the ageing, except for the collagen. The meat of animals slaughtered between 12 and 15 months of age showed attributes of quality independent of the genetic group and with seven days of ageing all animals had a desirable tenderness. / Mestre

Strip steaks from the McGregor genome project were used to evaluate the effects of sarcomere length, myofibrillar fragmentation index, 3 h postmortem pH, 24 h postmortem pH, marbling, electrical stimulation (ES), sarcoplasmic free calcium concentration, and collagen characteristics on tenderness as measured by Warner-Bratzler shear force (WBS). The WBS values were measured prior to this project so the animals were able to be separated into “tender” and “tough” groups using a WBS value of 30 N as the separating point, steaks with a WBS value less than 30 N being “tender” and the others being “tough”. It was found that ES sides had lower WBS values, however, “tough” steaks showed a greater response to ES than “tender” steaks. ES sides also had higher sarcoplasmic free calcium concentration and lower 3 h postmortem pH. Tenderness is best predicted by treatment (ES versus NON-ES), however, there is some efficacy in using total collagen and collagen solubility in conjunction with treatment.

Gelation properties of a salt extracted pea (Pisum sativum) protein isolate (PPIs) were evaluated with a goal of using this isolate as a meat extender. Microbial transglutaminase (MTG) was used to improve gelation of PPIs, muscle protein isolate (MPI) from chicken breast and the two combined. Gelation properties were evaluated using small amplitude oscillatory rheology and texture analysis. SDS-PAGE and differential scanning calorimetry were used to examine protein structure. Minimum gelation concentration for PPIs was 5%, lower than the 14% obtained for a commercial pea protein isolate (PPIc), possibly because the PPIc undergone denaturation whereas PPIs had not. Storage modulus (G') and loss modulus (G") increased with protein concentration and maximum gel strength for PPIs occurred at pH 4.0 in 0.3M NaCl. Higher or lower pH values affected protein charge and the potential for network formation. Higher salt concentrations resulted in increased denaturation temperatures, to a point where the proteins did not denature at the 95ºC temperature used for gel formation. When both heating and cooling rate were increased, gel strength decreased, though the cooling rate had a greater impact. Chaotropic salts enhanced gel strength, whereas non-chaotropic salts stabilized protein structure and decreased gel formation. Based on effects of guanidine hydrochloride, urea, propylene glycol, β-mercaptoethanol, dithiothreitol and N-ethylmaleimide, hydrophobic and electrostatic interaction and hydrogen bonds were involved in pea protein gel formation but disulfide bond contribution was minimal. Gels formed with MPI at concentrations as low as 0.5% and were strongest at 95ºC, higher than the ~ 65ºC normally used in meat processing. Good gels were formed at pH 6 with 0.6 to 1.2 M NaCl. Addition of MTG increased gel strength for PPIs, MPI, and a combination of the two. SDS-PAGE showed that bands in the 35~100kDa range became fainter with higher MTG levels but no new bands were found to provide direct evidence of interaction between muscle and pea proteins. Improved gel strength for the MPI/PPI mixture (3:1) containing MTG suggested that some crosslinking occurred. Higher heating temperatures and MTG addition led to the formation of MPI/PPI gel and demonstrated the potential for utilization of pea protein in muscle foods.

Congenital myopathies are a rare and heterogeneous group of diseases. They are primarily characterised by skeletal muscle weakness and disease-specific pathological features. They harshly limit ordinary life and in severe cases, these myopathies are associated with early death of the affected individuals. The congenital myopathies investigated in this thesis are nemaline myopathy and myofibrillar myopathy. These diseases are usually caused by missense mutations in genes encoding myofibrillar proteins, but the exact mechanisms by which the point mutations in these proteins cause the overall weakness remain mysterious. Hence, in this thesis two different nemaline myopathy-causing actin mutations and one myofibrillar myopathy-causing myosin-mutation found in both human patients and mouse models were used to investigate the cascades of molecular and cellular events leading to weakness. I performed a broad range of functional and structural experiments including skinned muscle fibre mechanics, small-angle X-ray scattering as well as immunoblotting and histochemical techniques. Interestingly, according to my results, point mutations in myosin and actin differently modify myosin binding to actin, cross-bridge formation and muscle fibre force production revealing divergent mechanisms, that is, gain versus loss of function (papers I, II and IV). In addition, one point mutation in actin appears to have muscle-specific effects.  The presence of that mutant protein in respiratory muscles, i.e. diaphragm, has indeed more damaging consequences on myofibrillar structure than in limb muscles complexifying the pathophysiological mechanisms (paper II). As numerous atrophic muscle fibres can be seen in congenital myopathies, I also considered this phenomenon as a contributing factor to weakness and characterised the underlying causes in presence of one actin mutation. My results highlighted a direct muscle-specific up-regulation of the ubiquitin-proteasome system (paper III). All together, my research work demonstrates that mutation- and muscle-specific mechanisms trigger the muscle weakness in congenital myopathies. This gives important insights into the pathophysiology of congenital myopathies and will undoubtedly help in designing future therapies.

Gelation properties of a salt extracted pea (Pisum sativum) protein isolate (PPIs) were evaluated with a goal of using this isolate as a meat extender. Microbial transglutaminase (MTG) was used to improve gelation of PPIs, muscle protein isolate (MPI) from chicken breast and the two combined. Gelation properties were evaluated using small amplitude oscillatory rheology and texture analysis. SDS-PAGE and differential scanning calorimetry were used to examine protein structure. Minimum gelation concentration for PPIs was 5%, lower than the 14% obtained for a commercial pea protein isolate (PPIc), possibly because the PPIc undergone denaturation whereas PPIs had not. Storage modulus (G') and loss modulus (G") increased with protein concentration and maximum gel strength for PPIs occurred at pH 4.0 in 0.3M NaCl. Higher or lower pH values affected protein charge and the potential for network formation. Higher salt concentrations resulted in increased denaturation temperatures, to a point where the proteins did not denature at the 95ºC temperature used for gel formation. When both heating and cooling rate were increased, gel strength decreased, though the cooling rate had a greater impact. Chaotropic salts enhanced gel strength, whereas non-chaotropic salts stabilized protein structure and decreased gel formation. Based on effects of guanidine hydrochloride, urea, propylene glycol, β-mercaptoethanol, dithiothreitol and N-ethylmaleimide, hydrophobic and electrostatic interaction and hydrogen bonds were involved in pea protein gel formation but disulfide bond contribution was minimal. Gels formed with MPI at concentrations as low as 0.5% and were strongest at 95ºC, higher than the ~ 65ºC normally used in meat processing. Good gels were formed at pH 6 with 0.6 to 1.2 M NaCl. Addition of MTG increased gel strength for PPIs, MPI, and a combination of the two. SDS-PAGE showed that bands in the 35~100kDa range became fainter with higher MTG levels but no new bands were found to provide direct evidence of interaction between muscle and pea proteins. Improved gel strength for the MPI/PPI mixture (3:1) containing MTG suggested that some crosslinking occurred. Higher heating temperatures and MTG addition led to the formation of MPI/PPI gel and demonstrated the potential for utilization of pea protein in muscle foods.

Resumo: O presente estudo teve como objetivos: 1) Estabelecer possíveis correlações entre as técnicas de força de cisalhamento e o índice de fragmentação miofibrilar para analise da maciez da carne 2) Identificar as proteínas miofibrilares do músculo Longíssimus dorsi de bovinos de corte, associado à maciez da carne; 3) Estudar o efeito da raça, idade do abate e tempo de maturação, sobre a força de cisalhamento, o índice de fragmentação miofibrilar e as proteínas Miofibrilares. Foram utilizados 30 animais da raça Nelore e 30 animais ½ Nelore ½ Aberdeen Angus, criados no modelo biológico Superprecoce abatios nas idades de 12 meses, e de rebanhos comerciais abatidos com 24 meses de idade. As amostras foram submetidas à análise da força de cisalhamento em um Warner-Bratzler Shear Force mecânico e a determinação do MFI. A separação das proteínas foi realizada em eletroforese 1D. Para avaliar os efeitos de grupos genéticos, período de maturação e de idade ao abate sobre a Força de cisalhamento, Índice de fragmentação miofibrilar e intensidade das bandas proteícas, foi utilizado o procedimento GLM (SAS), e posteriormente obtidas às correlações lineares simples de Pearson utilizando-se o procedimento CORR (SAS). Os valores de intensidade das bandas foram submetidas à hidrólise tripsínica e à espectrometria de massas, utilizando um espectrômetro do tipo eletrospray triplo/quadrupolo. A composição genética não influenciou a maciez da carne, para as diferentes idades para a força de cisalhamento e o índice de fragmentação miofibrilar. O período de maturação só influenciou na maciez da carne até os 7 dias de maturação. A força de cisalhamento e o índice de fragmentação miofibrilar apresentaram correlações significativas. Não foi observada diferença entre idade... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study has the objective to: 1) Establish possible correlations between the shear force techniques and the index of myofibril fragmentation to analyze the meet tenderness 2) Identify the myofibrillar proteins of Longissimus dorsi muscle in bovines of cut related to meat tenderness; 3) Study the effect of race, slaughter age and time of maturation in the shear force, the index of myofibrillar fragmentation and myofibrillar proteins. We used 30 animals Nelore breed and 30 animals ½ Nelore ½ Aberdeen Angus, raised in the biological Superprecoce production system that was slaugher with 12 months and commercial animals slaughered with 24 months of age. The samples was submitted to mechanic Warner-Bratzler mechanical Shear Force and MFI determination. The separation of the proteins was held in eletroforese 1D. To evaluate the effect of genetic groups, period of maturation and slaughtering age in the Shear Force, the index of myofibrillar fragmentation and the intensity of band proteins, it was used the GLM(SAS) procedure, and after that the simple linear correlations of Pearson with CORR (SAS) procedure. The values of band intensity were submitted to trypsinic hydrolysis and mass spectrometry, using eletrospray/quadrupole spectrometer. The genetic composition did not influence the meat tenderness, to the different ages to the shear force and myofibrillar fragmentation index. The maturation period only influenced in the meat tenderness 7 days of maturation. The shear force and myofibril fragmentation index had significant correlations. It was not observed difference between age and racial group in tenderness and in degradation of myofibrillar proteins. Only the 30kDa band presented high correlation (r=0,60) with 14 days of maturation period in both the races. This band was submitted to ESI-3Q-MS analysis... (Complete abstract click electronic access below) / Orientador: Paulo Roberto Rodrigues Ramos / Coorientador: Hellen Julie Laure / Banca: Lewis Joel Greene / Banca: Clarica Izumi / Banca: Danísio Prado Murani / Banca: Pedro de Magalhães Padilha / Doutor