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Hypertrophic cardiomyopathy (HCM) is a primary disease of the myocardium. 4-11% of HCM is caused by mutations in cardiac troponin T (cTnT) and 65% of them are within the tropomyosin (TM)-binding TNT1 domain. Two of the known mutational hotspots within TNT1 are in the N and C-terminal domains. Unlike the N-terminal domain; no high-resolution structure exists for the highly conserved C-terminal domain limiting both our ability to understand the functional role of this extended domain in myofilament activation and molecular mechanism(s) of HCM. The Δ160E mutation is an in-frame deletion of a glutamic acid residue at position 160 of cTnT. This TNT1 C-terminal mutation is associated with an especially poor prognosis. The Δ160E mutation is located in a putative "hinge region" immediately adjacent to the unstructured flexible linker connecting the TM-binding TNT1 domain to the Ca²⁺-sensitive TNT2 domain. Unwinding of this α-helical hinge may provide the flexibility necessary for thin filament function. Previous regulated in vitro motility assay (R-IVM) data showed mutation-induced impairment of weak actomyosin binding. Thus, we hypothesized that the Δ160E mutation repositions the flexible linker which impairs weak electrostatic binding and ultimately leads to severe cardiac remodeling. The goal of our studies is two-fold: 1) to gain high-resolution insight into the position of the cTnT linker with respect to the C-terminus of TM, and 2) to identify Δ160E-induced positional changes using Fluorescence Resonance Energy Transfer (FRET) in a fully reconstituted thin filament. To this end, residues in the middle and distal regions of the cTnT linker were sequentially cysteine-substituted (A168C, A177C, A192C and S198C) and labeled with the energy donor IAEDANS. The energy acceptor, DABMI was attached to cysteine 190 (C190) in the C-terminal region of TM and FRET measurements were obtained in the presence and absence of Ca²⁺ and myosin subfragment 1 (S1). An all-atom thin filament model in the Ca²⁺–on state was employed to predict the pathogenic effects of the Δ160E mutation on the structure and the dynamics of the cTnT linker region. Our data suggest that the linker domain runs alongside the C-terminus of TM and is differentially repositioned by calcium, myosin and the Δ160E mutation. The Δ160E mutation moves the linker closer to the C-terminus of TM. The in silico model supported this finding and demonstrated a mutation-induced decrease in linker flexibility. Moreover, the model predicted a pathogenic change in the orientation of the middle region of the linker and in the position of the Ca²⁺-sensitive TNT2 domain and the TM-binding TNT1 domain in response to Δ160E mutation. Collectively, our findings suggest that the Δ160E mutation-induced changes in the structure, position and dynamics of the linker region cause steric blocking of weak myosin binding sites on actin and subsequent impairment of contraction and disruption of sarcomeric integrity. These studies, for the first time, provided information regarding the role of the extended linker in both myofilament activation and disease.

A maciez é um importante atributo de qualidade para os consumidores de carne. No entanto, não há dados específicos do impacto da maciez da carne ovina de Santa Inês sobre a percepção dos consumidores brasileiros. Portanto, o objetivo foi verificar a avaliação do consumidor de carne ovina apresentando diferentes forças de cisalhamento. Foram utilizados oito cordeiros da raça Santa Inês, que receberam a mesma dieta de terminação. Estes animais tinham 5 a 6 meses de idade e pesavam de 30 a 35 kg quando foram abatidos e suas meias carcaças foram utilizadas para obtenção de 16 amostras de Longissimus dorsi (LD). Estas foram distribuídas aleatoriamente entre quatro diferentes processamentos pós-abate (PA). Os LD usados para a obtenção da Carne Endurecida (CE) foram retirados logo após o abate (0h) e as amostras para os demais tratamentos foram retiradas na desossa às 24h PA. O tratamento para CE consistia em embalar o LD e mergulhá-lo em água a 0° C por duas horas, e logo após congelá-la. Para a obtenção da Carne Fresca (CF), o LD foi retirado na desossa (24h PA), embalado e congelado. E alguns dos bifes obtidos às 24 horas foram embalados a vácuo e maturados a 1°C durante três ou sete dias, que compõem a condição de Carne Maturada (CM) pós-morte, CM3 e CM7, respectivamente. Os principais dados obtidos foram de comprimento do sarcômero (CS), força de cisalhamento (FC), índice de fragmentação miofibrilar (IFM), bem como a aceitação de maciez, suculência e qualidade global por parte de 50 consumidores não treinados, usando escala hedônica de nove pontos. O CS foi de 1,44 &mu;m para CE, significativamente (p<0,05) mais curto que observado para CF, CM3 e CM7, que não diferiram entre si, com valores de 1,84, 1,89, 1,85 &mu;m (SEM=0,09), respectivamente. A CE alcançou FC média de 7,2 kgf, significativamente (P<0,05) mais alta que os três valores similares de 3,3, 3,1, 2,4 (SEM=0,44) observados para CF, CM3 e CM7, respectivamente. Quanto à IFM, os processos pós-abate tiveram diferença entre CE e CM7 (p<0,05), com IFM de 76,2 e 130,5, enquanto a CF e CM3 não diferiram entre os processos com valores de 97,9; 115,6; (SEM= 11,41), respectivamente. As amostras de CE receberam pontuação significativamente mais baixa (P<0,01) para todos os quesitos avaliados na análise sensorial, sendo textura 5,43, suculência 6,51 e qualidade global 5,92, os demais tratamentos tiveram notas acima de 7 para todas as características, mas não diferiram entre si (P<0,01). Os consumidores foram capazes de perceber e avaliaram negativamente a maciez da carne de cordeiro que apresentou alta força de cisalhamento quando comparada com carnes com valores que as caracterizariam como macias. As avaliações de suculência e qualidade global acompanharam a percepção da maciez. Concluiu-se que os consumidores percebem e avaliam negativamente carne de cordeiro com alto valor de força de cisalhamento. No entanto, os dados obtidos não foram capazes de elucidar impacto de diferenças menores na força de cisalhamento entre amostras duras e macias sobre a aceitação do consumidor não treinado. / Tenderness is an important meat quality trait for consumers. However, there is no specific data in the impact of Santa Inês lamb meat tenderness on Brazilian consumers perception. Therefore, objective was to verify the consumer evaluation of lamb meat with different shear force. We used eight Santa Inês lambs that were fed the same diet. These animals of 5 to 6 months of age and weighing 30 to 35 Kg were slaughtered and their half carcasses were used to obtain 16 samples of Longissimus dorsi (LD). These were randomly assigned among four diferent post-slaughter (PS) managements. The LD used for obtaining Toughened Meat (TM) were removed immediately after slaughter (0h ps) and samples for the other managements were obtained from boning at 24h PS. The samples from TM consisted in wrapping excised LD in plastic bag and dipping it into water bath with ice at 0°C for two hours, followed by freezing. Fresh Meat (FM) was obtained from LD removed at boning (24 hours postmortem), followed by wrapping and freezing. And some of steaks obtained at 24 hours were vaccumm packaged and aged at 1 °C for three or seven days, which compose the aging (A) postmortem, A3 and A7, respectively. The sarcomere length (SL), shear force (SF), myofibrillar fragmentation index (MFI), as well as sensory analysis of tenderness, juiciness and overall quality acceptance by 50 not trained consumers using hedonic scale of nine points, were obtained. The SL was 1.44 &mu;m for TM, significantly (P<0.05) shorter than the other treatments. The values of 1.84, 1.89, 1.85 (SEM=0,09) &mu;m for FM, A3 and A7, respectively, didn\'t differ and are values normally reported for post-rigor lamb meat. MT reached an average 7.2 kgf for SF, significantly (P <0.05) higher than 3.3, 3.1, 2.4 (SEM= 0,44) observed for FM, A7 and A3, respectively. As for the MFI, post-slaughter processes had difference between TM and A7 (P<0.05), with IFM 76.2 and 130.5, while FM and A3 did not differ between cases with values 97.9; 115.6; (SEM = 11.41), respectively. Samples from TM received the lowest scores (P<0.01) for all items assessed in sensory analysis, and texture 5.43, juiciness 6.51 and overall quality 5.92, the other treatments were notes above 7 for all traits, but not significantly different (P<0.01). Consumers were able to understand and evaluate negatively the tenderness of lamb that showed high shear force compared to meat with values at low levels, which characterize as tender. It was concluded that consumers will perceive and evaluate negatively lamb meat with high shear force value. However, the results are not conclusive whether smaller differences between tough and tender meat would still have impact on not trained consumer acceptance.

O hormônio tireoideano (T3) é um potente regulador das funções cardíacas e musculares esqueléticas. Desse modo, o presente trabalho identificou o efeito de tal hormônio nas proteínas da banda M sarcomérica, as quais fazem parte das proteínas estruturais cardíacas e musculares esqueléticas. O T3 diminuiu a expressão da proteína M no coração, uma das proteínas da banda M, e agiu diretamente no gene dessa proteína M. No músculo esquelético, T3 aumentou a expressão de EH-miomesina no músculo sóleo e reduziu a expressão de proteína M no músculo extensor digital longo (EDL). Portanto, pode-se concluir que o T3 possui uma importante função na regulação da expressão de proteínas estruturais musculares e a ausência dessas proteínas pode ocasionar lesões das estruturas musculares cardíacas e esqueléticas. / Thyroid hormone (T3) regulates many functions of the heart and skeletal muscle. In this way, the present work identified the effect of T3 on the sarcomeric M band proteins, which are structural proteins from the heart and skeletal muscle. T3 down regulated M protein expression in the heart, one of the M band proteins, and, moreover, T3 could regulate M protein gene directly. In the skeletal muscle, T3 up regulated EH-myomesin expression in soleus muscle and T3 down regulated M protein expression in the extensor digitorum longus muscle (EDL). Therefore, we could conclude that T3 has an essential function in the regulation of muscle structural protein and the absence of these proteins could cause lesions at cardiac and skeletal muscle structures.

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.

The assembly, form, and function of the heart is regulated by complex mechanical signals originating from intrinsic and extrinsic sources, such as the cytoskeleton and the extracellular matrix. During development, mechanical forces influence the self-assembly of highly organized ventricular myocardium. However, mechanical overload induces maladaptive remodeling of tissue structure and eventual failure. Thus, mechanical forces potentiate physiological or pathological remodeling, depending on factors such as frequency and magnitude. We hypothesized that mechanical stimuli in the form of microenvironmental stiffness, cytoskeletal architecture, or cyclic stretch regulate cell-cell junction formation and cytoskeletal remodeling during development and disease. To test this, we engineered cardiac tissues in vitro and quantified structural and functional remodeling over multiple spatial scales in response to diverse mechanical perturbations mimicking development and disease. We first asked if the mechanical microenvironment impacts tissue assembly. To investigate this, we cultured two-cell cardiac µtissues on flexible substrates with tunable stiffness and monitored cell-cell junction formation over time. As myocytes transitioned from isolated cells to interconnected µtissues, focal adhesions disassembled near cell-cell interfaces and mechanical forces were transmitted almost completely through cell-cell junctions. However, µtissues cultured on stiff substrates mimicking fibrotic microenvironments retained focal adhesions near the cell-cell interface, potentially to reinforce the cell-cell junction in response to excessive forces generated by myofibrils in stiff microenvironments. Intercellular electrical conductance between myocytes was measured as a function of connexin 43 immunosignal and the length-to-width ratio of cell pairs. We observed that conductance was correlated to connexin 43 immunosignal and cell pair length-to-width ratio, indicating that tissue architecture can affect electrical coupling. The impact of mechanical overload was also determined by applying chronic cyclic stretch to engineered cardiac tissues. Stretch activated gene expression patterns characteristic of pathological remodeling, including up-regulation of focal adhesion genes, and impacted sarcomere alignment and myocyte shape. Furthermore, chronic cyclic stretch altered intracellular calcium cycling in a manner similar to heart failure and decreased contractile stress generation, suggestive of maladaptive remodeling. In summary, we show that the assembly, form, and function of cardiac tissue is sensitive to a wide range of mechanical cues that emerge during physiological and pathological growth. / Engineering and Applied Sciences

Having an in vivo model of development that develops quickly and efficiently is important for investigators to elucidate the critical steps, components and signaling pathways involved in building a myofibril; hence a compliant in vivo model would provide a pivotal foundation for deciphering muscle disease mechanisms as well as the development of myopathy-related therapeutics. Here, we take advantage of a relatively quick, cost effective, and molecularly pliable developmental model system in the Xenopus laevis (frog) embryo and establish it as an in vivo model to study the roles of sarcomeric proteins during de novo myofibrillogenesis.Using the Xenopus model, we elucidated the functional roles of Leiomodin3 (Lmod3) and Tropomodulin 4 (Tmod4) during de novo skeletal myofibrillogenesis. Tmods have been demonstrated to contribute to thin filament length uniformity by regulating both elongation and depolymerization of actin-thin filaments' pointed-ends. Lmods, which are structurally related to Tmod proteins also localize to actin filament pointed-ends. In situ hybridization studies demonstrated that of their respective families, only tmod4 and lmod3 transcripts were expressed at high levels in skeletal muscle from the earliest stages of development. When reducing their protein levels via morpholino (MO) treatment, thin filament regulation and sarcomere assembly were compromised. Surprisingly, alternate rescues (i.e., lmod3 mRNA co-injected with Tmod4 MO and vice versa) partially restored myofibril structure and actin-thin filament organization. Thus, our results not only indicate that both Tmod4 and Lmod3 are critical for myofibrillogenesis during Xenopus skeletal muscle development, but also revealed that they may share redundant functions during skeletal muscle thin filament assembly.

Biochemical changes in muscle postmortem have been associated with initial beef tenderness early postmortem, and with improvements in tenderness during postmortem storage, defined as meat aging. Differences in the initial contractile state of the sarcomere, the ionic environment of the sarcoplasm including pH, the activity of neutral proteolytic enzymes, and collagen content and solubility have been associated with beef tenderness. In Phase I, steaks from four genetic lines of steers and heifers were used to understand the biochemical differences between tough and tender steaks. The most tender (< 30 N Warner Bratzler shear force (WBS)) and toughest Longissimus steaks (< 30 N WBS) from Angus, Braford, Brangus, and Simbrah heifers and steers were used. For Phase II, samples were obtained from a subset of Santa Cruz yearling heifers selected based of genotypes for tenderness (tough and tender) using a commercial genetic marker. Within genotype for tenderness, each animal was randomly assigned to one of four growth enhancement treatments. The most tender (< 30 N WBS) and toughest Longissimus steaks (< 30 N WBS) were selected for use in this study. In Phase I, tough steaks after 3, 10, and 17d postmortem had higher (P < 0.0005) WBS values than tender steaks. Tender steaks came from carcass with slightly higher (P = 0.008) marbling score and (P = 0.01) Quality grade. Sarcomere length, total and soluble collagen, potassium concentration, and m and µcalpain did not differ (P > 0.05) between tough and tender steaks. Sodium concentration at 10 d was higher (P = 0.03) in tough steaks, but only account for 0.05% of the variation in WBS at 3d. Tender steaks had less (P = 0.04) intact desmin at 24h, but intact desmin was not correlated (P > 0.05) with WBS. In Phase II, tough steaks after 3, 10, and 17d postmortem had higher (P < 0.0001) WBS values than tender steaks. Tender steaks came from carcass with slightly higher (P < 0.03) marbling score and (P = 0.02) Quality grade. Tender teaks were slightly lighter (P = 0.02), with more red (P = 0.02) and yellow (P = 0.007) color, and had slightly lower (P = 0.02) pH, compared with tough steaks. Sarcomere length, total and soluble collagen, sodium and potassium concentration, and m and µcalpain did not differ (P > 0.05) between tough and tender steaks. Tender steaks had less (P < 0.0001) intact desmin at 17d postmortem than tough steaks. Intact desmin at 17d was responsible for 4%, 47%, and 30% of WBS variation after 3, 10, and 17d postmortem, respectively. The slight difference in marbling and quality grade did not account for a significant amount of variation in WBS. However, meat color and pH accounted for variation in shear WBS. Calcium flux may have influenced meat tenderness by activation of calpains and may have altered protein to protein interactions. Results suggested that marbling, µ calpain activity, and desmin degradation, and to a lesser extent pH and meat color contributed to meat tenderness.

A maciez é um importante atributo de qualidade para os consumidores de carne. No entanto, não há dados específicos do impacto da maciez da carne ovina de Santa Inês sobre a percepção dos consumidores brasileiros. Portanto, o objetivo foi verificar a avaliação do consumidor de carne ovina apresentando diferentes forças de cisalhamento. Foram utilizados oito cordeiros da raça Santa Inês, que receberam a mesma dieta de terminação. Estes animais tinham 5 a 6 meses de idade e pesavam de 30 a 35 kg quando foram abatidos e suas meias carcaças foram utilizadas para obtenção de 16 amostras de Longissimus dorsi (LD). Estas foram distribuídas aleatoriamente entre quatro diferentes processamentos pós-abate (PA). Os LD usados para a obtenção da Carne Endurecida (CE) foram retirados logo após o abate (0h) e as amostras para os demais tratamentos foram retiradas na desossa às 24h PA. O tratamento para CE consistia em embalar o LD e mergulhá-lo em água a 0° C por duas horas, e logo após congelá-la. Para a obtenção da Carne Fresca (CF), o LD foi retirado na desossa (24h PA), embalado e congelado. E alguns dos bifes obtidos às 24 horas foram embalados a vácuo e maturados a 1°C durante três ou sete dias, que compõem a condição de Carne Maturada (CM) pós-morte, CM3 e CM7, respectivamente. Os principais dados obtidos foram de comprimento do sarcômero (CS), força de cisalhamento (FC), índice de fragmentação miofibrilar (IFM), bem como a aceitação de maciez, suculência e qualidade global por parte de 50 consumidores não treinados, usando escala hedônica de nove pontos. O CS foi de 1,44 &mu;m para CE, significativamente (p<0,05) mais curto que observado para CF, CM3 e CM7, que não diferiram entre si, com valores de 1,84, 1,89, 1,85 &mu;m (SEM=0,09), respectivamente. A CE alcançou FC média de 7,2 kgf, significativamente (P<0,05) mais alta que os três valores similares de 3,3, 3,1, 2,4 (SEM=0,44) observados para CF, CM3 e CM7, respectivamente. Quanto à IFM, os processos pós-abate tiveram diferença entre CE e CM7 (p<0,05), com IFM de 76,2 e 130,5, enquanto a CF e CM3 não diferiram entre os processos com valores de 97,9; 115,6; (SEM= 11,41), respectivamente. As amostras de CE receberam pontuação significativamente mais baixa (P<0,01) para todos os quesitos avaliados na análise sensorial, sendo textura 5,43, suculência 6,51 e qualidade global 5,92, os demais tratamentos tiveram notas acima de 7 para todas as características, mas não diferiram entre si (P<0,01). Os consumidores foram capazes de perceber e avaliaram negativamente a maciez da carne de cordeiro que apresentou alta força de cisalhamento quando comparada com carnes com valores que as caracterizariam como macias. As avaliações de suculência e qualidade global acompanharam a percepção da maciez. Concluiu-se que os consumidores percebem e avaliam negativamente carne de cordeiro com alto valor de força de cisalhamento. No entanto, os dados obtidos não foram capazes de elucidar impacto de diferenças menores na força de cisalhamento entre amostras duras e macias sobre a aceitação do consumidor não treinado. / Tenderness is an important meat quality trait for consumers. However, there is no specific data in the impact of Santa Inês lamb meat tenderness on Brazilian consumers perception. Therefore, objective was to verify the consumer evaluation of lamb meat with different shear force. We used eight Santa Inês lambs that were fed the same diet. These animals of 5 to 6 months of age and weighing 30 to 35 Kg were slaughtered and their half carcasses were used to obtain 16 samples of Longissimus dorsi (LD). These were randomly assigned among four diferent post-slaughter (PS) managements. The LD used for obtaining Toughened Meat (TM) were removed immediately after slaughter (0h ps) and samples for the other managements were obtained from boning at 24h PS. The samples from TM consisted in wrapping excised LD in plastic bag and dipping it into water bath with ice at 0°C for two hours, followed by freezing. Fresh Meat (FM) was obtained from LD removed at boning (24 hours postmortem), followed by wrapping and freezing. And some of steaks obtained at 24 hours were vaccumm packaged and aged at 1 °C for three or seven days, which compose the aging (A) postmortem, A3 and A7, respectively. The sarcomere length (SL), shear force (SF), myofibrillar fragmentation index (MFI), as well as sensory analysis of tenderness, juiciness and overall quality acceptance by 50 not trained consumers using hedonic scale of nine points, were obtained. The SL was 1.44 &mu;m for TM, significantly (P<0.05) shorter than the other treatments. The values of 1.84, 1.89, 1.85 (SEM=0,09) &mu;m for FM, A3 and A7, respectively, didn\'t differ and are values normally reported for post-rigor lamb meat. MT reached an average 7.2 kgf for SF, significantly (P <0.05) higher than 3.3, 3.1, 2.4 (SEM= 0,44) observed for FM, A7 and A3, respectively. As for the MFI, post-slaughter processes had difference between TM and A7 (P<0.05), with IFM 76.2 and 130.5, while FM and A3 did not differ between cases with values 97.9; 115.6; (SEM = 11.41), respectively. Samples from TM received the lowest scores (P<0.01) for all items assessed in sensory analysis, and texture 5.43, juiciness 6.51 and overall quality 5.92, the other treatments were notes above 7 for all traits, but not significantly different (P<0.01). Consumers were able to understand and evaluate negatively the tenderness of lamb that showed high shear force compared to meat with values at low levels, which characterize as tender. It was concluded that consumers will perceive and evaluate negatively lamb meat with high shear force value. However, the results are not conclusive whether smaller differences between tough and tender meat would still have impact on not trained consumer acceptance.

O hormônio tireoideano (T3) é um potente regulador das funções cardíacas e musculares esqueléticas. Desse modo, o presente trabalho identificou o efeito de tal hormônio nas proteínas da banda M sarcomérica, as quais fazem parte das proteínas estruturais cardíacas e musculares esqueléticas. O T3 diminuiu a expressão da proteína M no coração, uma das proteínas da banda M, e agiu diretamente no gene dessa proteína M. No músculo esquelético, T3 aumentou a expressão de EH-miomesina no músculo sóleo e reduziu a expressão de proteína M no músculo extensor digital longo (EDL). Portanto, pode-se concluir que o T3 possui uma importante função na regulação da expressão de proteínas estruturais musculares e a ausência dessas proteínas pode ocasionar lesões das estruturas musculares cardíacas e esqueléticas. / Thyroid hormone (T3) regulates many functions of the heart and skeletal muscle. In this way, the present work identified the effect of T3 on the sarcomeric M band proteins, which are structural proteins from the heart and skeletal muscle. T3 down regulated M protein expression in the heart, one of the M band proteins, and, moreover, T3 could regulate M protein gene directly. In the skeletal muscle, T3 up regulated EH-myomesin expression in soleus muscle and T3 down regulated M protein expression in the extensor digitorum longus muscle (EDL). Therefore, we could conclude that T3 has an essential function in the regulation of muscle structural protein and the absence of these proteins could cause lesions at cardiac and skeletal muscle structures.