Cardiac troponin T N-domain variant destabilizes the actin interface resulting in disturbed myofilament function.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament's relaxed state resulting in an increased number of crossbridges during Ca2+ activation. Additionally, in the low Ca2+-relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca2+, and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.

Etiology of genetic muscle disorders induced by mutations in fast and slow skeletal MyBP-C paralogs.

Skeletal muscle, a highly complex muscle type in the eukaryotic system, is characterized by different muscle subtypes and functions associated with specific myosin isoforms. As a result, skeletal muscle is the target of numerous diseases, including distal arthrogryposes (DAs). Clinically, DAs are a distinct disorder characterized by variation in the presence of contractures in two or more distal limb joints without neurological issues. DAs are inherited, and up to 40% of patients with this condition have mutations in genes that encode sarcomeric protein, including myosin heavy chains, troponins, and tropomyosin, as well as myosin binding protein-C (MYBPC). Our research group and others are actively studying the specific role of MYBPC in skeletal muscles. The MYBPC family of proteins plays a critical role in the contraction of striated muscles. More specifically, three paralogs of the MYBPC gene exist, and these are named after their predominant expression in slow-skeletal, fast-skeletal, and cardiac muscle as sMyBP-C, fMyBP-C, and cMyBP-C, respectively, and encoded by the MYBPC1, MYBPC2, and MYBPC3 genes, respectively. Although the physiology of various types of skeletal muscle diseases is well defined, the molecular mechanism underlying the pathological regulation of DAs remains to be elucidated. In this review article, we aim to highlight recent discoveries involving the role of skeletal muscle-specific sMyBP-C and fMyBP-C as well as their expression profile, localization in the sarcomere, and potential role(s) in regulating muscle contractility. Thus, this review provides an overall summary of MYBPC skeletal paralogs, their potential roles in skeletal muscle function, and future research directions.

Arrhythmogenic Cardiomyopathy: Exercise Pitfalls, Role of Connexin-43, and Moving beyond Antiarrhythmics.

Arrhythmogenic Cardiomyopathy (ACM), a Mendelian disorder that can affect both left and right ventricles, is most often associated with pathogenic desmosomal variants that can lead to fibrofatty replacement of the myocardium, a pathological hallmark of this disease. Current therapies are aimed to prevent the worsening of disease phenotypes and sudden cardiac death (SCD). Despite the use of implantable cardioverter defibrillators (ICDs) there is no present therapy that would mitigate the loss in electrical signal and propagation by these fibrofatty barriers. Recent studies have shown the influence of forced vs. voluntary exercise in a variety of healthy and diseased mice; more specifically, that exercised mice show increased Connexin-43 (Cx43) expression levels. Fascinatingly, increased Cx43 expression ameliorated the abnormal electrical signal conduction in the myocardium of diseased mice. These findings point to a major translational pitfall in current therapeutics for ACM patients, who are advised to completely cease exercising and already demonstrate reduced Cx43 levels at the myocyte intercalated disc. Considering cardiac dysfunction in ACM arises from the loss of cardiomyocytes and electrical signal conduction abnormalities, an increase in Cx43 expression-promoted by low to moderate intensity exercise and/or gene therapy-could very well improve cardiac function in ACM patients.

Fast skeletal myosin-binding protein-C regulates fast skeletal muscle contraction.

Fast skeletal myosin-binding protein-C (fMyBP-C) is one of three MyBP-C paralogs and is predominantly expressed in fast skeletal muscle. Mutations in the gene that encodes fMyBP-C, MYBPC2, are associated with distal arthrogryposis, while loss of fMyBP-C protein is associated with diseased muscle. However, the functional and structural roles of fMyBP-C in skeletal muscle remain unclear. To address this gap, we generated a homozygous fMyBP-C knockout mouse (C2-/-) and characterized it both in vivo and in vitro compared to wild-type mice. Ablation of fMyBP-C was benign in terms of muscle weight, fiber type, cross-sectional area, and sarcomere ultrastructure. However, grip strength and plantar flexor muscle strength were significantly decreased in C2-/- mice. Peak isometric tetanic force and isotonic speed of contraction were significantly reduced in isolated extensor digitorum longus (EDL) from C2-/- mice. Small-angle X-ray diffraction of C2-/- EDL muscle showed significantly increased equatorial intensity ratio during contraction, indicating a greater shift of myosin heads toward actin, while MLL4 layer line intensity was decreased at rest, indicating less ordered myosin heads. Interfilament lattice spacing increased significantly in C2-/- EDL muscle. Consistent with these findings, we observed a significant reduction of steady-state isometric force during Ca2+-activation, decreased myofilament calcium sensitivity, and sinusoidal stiffness in skinned EDL muscle fibers from C2-/- mice. Finally, C2-/- muscles displayed disruption of inflammatory and regenerative pathways, along with increased muscle damage upon mechanical overload. Together, our data suggest that fMyBP-C is essential for maximal speed and force of contraction, sarcomere integrity, and calcium sensitivity in fast-twitch muscle.

Basic residues within the cardiac troponin T C terminus are required for full inhibition of muscle contraction and limit activation by calcium.

Striated muscle is activated by myosin- and actin-linked processes, with the latter being regulated through changes in the position of tropomyosin relative to the actin surface. The C-terminal region of cardiac troponin T (TnT), a tropomyosin-associated protein, is required for full TnT inactivation at low Ca2+ and for limiting its activation at saturating Ca2+ Here, we investigated whether basic residues in this TnT region are involved in these activities, whether the TnT C terminus undergoes Ca2+-dependent conformational changes, and whether these residues affect cardiac muscle contraction. We generated a human cardiac TnT variant in which we replaced seven C-terminal Lys and Arg residues with Ala and added a Cys residue at either position 289 or 275 to affix a fluorescent probe. At pCa 3.7, actin filaments containing high-alanine TnT had an elevated ATPase rate like that obtained when the last TnT 14 residues were deleted. Acrylodan-tropomyosin fluorescence changes and S1-actin binding kinetics revealed that at pCa 8, the high-alanine TnT-containing filaments did not enter the first inactive state. FRET analyses indicated that the C-terminal TnT region approached Cys-190 of tropomyosin as actin filaments transitioned to the inactive B state; that transition was abolished with high-alanine TnT. High-alanine TnT-containing cardiac muscle preparations had increased Ca2+ sensitivity of both steady-state isometric force and sinusoidal stiffness as well as increased maximum steady-state isometric force and sinusoidal stiffness. We conclude that C-terminal basic residues in cardiac TnT are critical for the regulation of cardiac muscle contraction.

Troponin through the looking-glass: emerging roles beyond regulation of striated muscle contraction.

Troponin is a heterotrimeric Ca2+-binding protein that has a well-established role in regulating striated muscle contraction. However, mounting evidence points to novel cellular functions of troponin, with profound implications in cancer, cardiomyopathy pathogenesis and skeletal muscle aging. Here, we highlight the non-canonical roles and aberrant expression patterns of troponin beyond the sarcomeric milieu. Utilizing bioinformatics tools and online databases, we also provide pathway, subcellular localization, and protein-protein/DNA interaction analyses that support a role for troponin in multiple subcellular compartments. This emerging knowledge challenges the conventional view of troponin as a sarcomere-specific protein exclusively involved in muscle contraction and may transform the way we think about sarcomeric proteins, particularly in the context of human disease and aging.

Temporomandibular joint disorders as the only manifestation of juvenile idiopathic arthritis: a case report / Comprometimento da articulação temporomandibular como única manifestação da artrite idiopática juvenil: um relato de caso

ABSTRACT Juvenile idiopathic arthritis is a term used to include all chronic childhood arthritis of unknown etiology. It is characterized by chronic inflammation persisting for at least 6 weeks, beginning before 16 years of age. The characteristics present are chronic synovitis, arthralgia, impaired joint mobility in at least one joint, and erosion with destruction of cartilage and subchondral bone, that could be associated or not with systemic involvement, according to each subtype of the disease. During the pathologic process, the temporomandibular joint can be involved by the juvenile idiopathic arthritis, resulting in severe mandibular dysfunction, with higher frequency in female patients. Initially, these lesions can show minor alterations like flattening of the condyle, erosions, and evolve to severe lesions, like destruction of the head of the condyle. We report a case of male patient who had destruction of both condyles, as a result from juvenile idiopathic arthritis. Proposed mechanisms to explain the juvenile idiopathic arthritis was reviewed. In this report the patient did not have pain or inflammatory process, and the temporomandibular diseases was the only manifestation.

RESUMO Artrite idiopática juvenil é um termo usado para incluir toda artrite infantil crônica de etiologia desconhecida. É caracterizada por uma inflamação crônica, que persiste por pelo menos 6 semanas, com início antes dos 16 anos de idade. As características presentes são sinovite crônica, artralgia, mobilidade articular diminuída em pelo menos uma articulação, e erosão com destruição da cartilagem e do osso subcondral, podendo ser associada ou não com o envolvimento sistêmico, de acordo com cada subtipo da doença. Durante o processo patológico, a articulação temporomandibular pode ser envolvida pela artrite idiopática juvenil, resultando em disfunção mandibular severa, com maior frequência em pacientes do sexo feminino. Inicialmente, estas lesões podem mostrar pequenas alterações, como achatamento do côndilo e erosões, e evoluir para lesões graves, como a destruição da cabeça do côndilo. Relatou-se o caso de um paciente do sexo masculino, que apresentou destruição de ambos os côndilos, como resultado da artrite idiopática juvenil. Os mecanismos para explicar a artrite idiopática juvenil foram revisados na literatura. Neste relato de caso, o paciente não apresentou dor e nem processo inflamatório, sendo o comprometimento da articulação temporomandibular a única manifestação.

S-Nitrosylation of Sarcomeric Proteins Depresses Myofilament Ca2+)Sensitivity in Intact Cardiomyocytes.

AIMS: The heart responds to physiological and pathophysiological stress factors by increasing its production of nitric oxide (NO), which reacts with intracellular glutathione to form S-nitrosoglutathione (GSNO), a protein S-nitrosylating agent. Although S-nitrosylation protects some cardiac proteins against oxidative stress, direct effects on myofilament performance are unknown. We hypothesize that S-nitrosylation of sarcomeric proteins will modulate the performance of cardiac myofilaments. RESULTS: Incubation of intact mouse cardiomyocytes with S-nitrosocysteine (CysNO, a cell-permeable low-molecular-weight nitrosothiol) significantly decreased myofilament Ca(2+) sensitivity. In demembranated (skinned) fibers, S-nitrosylation with 1 µM GSNO also decreased Ca(2+) sensitivity of contraction and 10 µM reduced maximal isometric force, while inhibition of relaxation and myofibrillar ATPase required higher concentrations (≥ 100 µM). Reducing S-nitrosylation with ascorbate partially reversed the effects on Ca(2+) sensitivity and ATPase activity. In live cardiomyocytes treated with CysNO, resin-assisted capture of S-nitrosylated protein thiols was combined with label-free liquid chromatography-tandem mass spectrometry to quantify S-nitrosylation and determine the susceptible cysteine sites on myosin, actin, myosin-binding protein C, troponin C and I, tropomyosin, and titin. The ability of sarcomere proteins to form S-NO from 10-500 µM CysNO in intact cardiomyocytes was further determined by immunoblot, with actin, myosin, myosin-binding protein C, and troponin C being the more susceptible sarcomeric proteins. INNOVATION AND CONCLUSIONS: Thus, specific physiological effects are associated with S-nitrosylation of a limited number of cysteine residues in sarcomeric proteins, which also offer potential targets for interventions in pathophysiological situations.

Análise comparativa entre tipos de preparo para facetas laminadas, em relação à resistência da cerâmica, em ensaios de impacto / Comparative analysis between laminate veneer preparation types regarding ceramic resistance upon impact mechanical impact tests

Objetivo: este estudo avaliou a resistência à fratura de facetas confeccionadas sobre dois tipos de preparo. Métodos: utilizando-se dentes bovinos, dois grupos foram preparados: grupo I (redução incisal de 1 mm) e grupo II (redução incisal de 3 mm). Facetas laminadas foram enceradas sobre preparos dentais e confeccionadas em cerâmica IPS e.max (Ivoclar Vivadent). As superfícies internas das facetas foram tratadas com ácido fluorídrico a 10% durante 20 segundos e silanizadas. A superfície preparada do dente foi condicionada durante 15 segundos com ácido fosfórico a 37%, lavada abundantemente e secada levemente. O adesivo foi aplicado em duas camadas: dente e lado interno do laminado. A cimentação foi realizada com cimento resinoso fotopolimerizável sob pressão digital, e uma máquina de teste tipo pendular foi utilizada para simular o impacto no corpo-de-prova. Resultados: os dados de energia absorvida na fratura pelas facetas laminadas foram submetidos ao teste t de Student para amostras independentes (α = 5%), que demonstrou não haver diferença significativa entre os grupos com redução incisal de 1 mm e redução incisal de 3 mm (p=0,724). Observou-se, no grupo I, 10% das facetas laminadas com perda total, 30% extensa, mas parcial, e 60% parcial. Já no grupo II, 0% das facetas laminadas com perda total, 10% extensa, mas parcial, e 90% parcial. Conclusão: embora não tenha sido demonstradas diferenças na energia absorvida entre os dois tipos de preparo, o modo de fratura nos preparos com redução incisal de 3 mm foi mais favorável.

Objective: to evaluate the fracture resistance of laminate veneers on two preparation types. Methods: two groups were made based on bovine teeth: group I (1 mm incisal reduction) and group II (3 mm incisal reduction). All Laminate veneers were waxed over tooth preparations and fabricated with the IPS e.max ceramic (Ivoclar Vivadent). Their inner surfaces were treated with 10% hydrofluoric acid for 20 seconds and received a silane agent. The tooth surface was conditioned with 37% phosphoric acid, washed, and slightly dried. The adhesive was applied at the tooth and the laminate veneer. A photo-cured luting agent was used under fi nger pressure, and the samples were placed in an impact machine test. The fracture energy data was submitted to the Student´s t test for independent samples (5% significance level). Results: no statistical difference was observed (p = 0.724). At group I, 10% of the laminates demonstrated complete loss, 30% extensive but partial loss, and 60% partial loss. For group II, the figures were 0% for complete loss, 10% extensive but partial, and 90% partial. Conclusion: although no diff erences were seen for the absorbed energy, the 3 mm incisal reduction design was the most favorable.

Effect of the addition of propolis on a soft denture liner on bond strength with an acrylic resin / Efeito da adição de própolis em um reembasador resiliente de prótese na resistência de união com resina acrílica

Objective ­ To evaluate the effect on bond strength of an acrylic resin and thermoatived resilient denture liner material with different concentrations of propolis. Methods ­ Fourty eight specimens of acrylic resin (Lucitone 550, Dentsply) were made and united with the soft liner (Coe Soft, GC America) with different concentrations of propolis: group 1 (control) ­ only Coe Soft; Group 2 ­ Coe Soft + 75 mg of propolis; Group 3 ­ Coe Soft + 150 mg of propolis, and Group 4 ­ Coe Soft + 300 mg of propolis. The union traction resistance test was performed, and the type of fracture was evaluated. The data were analyzed descriptively and by one-way ANOVA, followed by Tukey's test (p=0.05). Results ­ There was no difference between the control group and when 75 mg of propolis was added (p>0.05), while the group with 150 mg of propolis showed lower bond strength (p<0.05), However, group 4 did not differ from the others (p>0.05). Regarding the kind of fails, the control group and group 2 showed the same failures: 91.7% cohesive and 8.3% adhesive. Group 3 showed only cohesive failure, and group 4 had cohesive failures (58.4%), adhesive (8.3%) and mixed (33.3%). Conclusions ­ It can be concluded that 75 mg of propolis extract may be considered the most suitable concentration to be added in the soft denture liner, considering that the properties of soft denture liner was not changed.

Objetivo ­ Avaliar o efeito na resistência de união entre resina acrílica e reembasador resiliente de prótese termoativado com diferentes concentrações de própolis. Métodos ­ Quarenta e oito espécimes de resina acrílica (Lucitone 550, Dentsply) foram confeccionados e unidos ao reembasador resiliente (Coe Soft, GC América) com diferentes concentrações de própolis: Grupo 1 (controle) ­ apenas o Coe Soft, Grupo 2 ­ Coe Soft + 75 mg de própolis; Grupo 3 ­ Coe Soft + 150 de própolis e Grupo 4 ­ Coe Soft + 300 mg de própolis. Foi realizado o teste de resistência à tração e o tipo de fratura foi avaliado. Os dados foram analisados de forma descritiva e através de análise de variância a um critério, seguido pelo teste de Tukey (p=0,05). Resultado ­ Não houve diferença entre o grupo controle e quando 75 mg de própolis foi adicionao (p>0,05), enquanto o grupo com 150 mg de própolis mostrou menor resistência de união (p<0,05). No entanto, o grupo 4 não diferiu dos demais (p>0,05). Em relação ao tipo de falha, o grupo controle e o grupo 2 apresentaram as mesmas falhas: 91,7% coesivas e 8,3% adesivas. O grupo 3 obteve apenas falha coesiva, e o grupo 4 teve falhas coesivas (58,4%), adesivas (8,3%) e mistas (33,3%). Conclusões ­ Conclui-se que 75mg de extrato de própolis pode ser considerada a concentração mais adequada para ser adicionada ao reembasador de prótese, considerando-se que as propriedades deste reembasador resiliente de prótese não foram alteradas.

Pathogenesis associated with a restrictive cardiomyopathy mutant in cardiac troponin T is due to reduced protein stability and greatly increased myofilament Ca2+ sensitivity.

BACKGROUND: Dilated and hypertrophic cardiomyopathy mutations in troponin can blunt effects of protein kinase A (PKA) phosphorylation of cardiac troponin I (cTnI), decreasing myofilament Ca2+-sensitivity; however this effect has never been tested for restrictive cardiomyopathy (RCM) mutants. This study explores whether an RCM cardiac troponin T mutant (cTnT-ΔE96) interferes with convergent PKA regulation and if TnT instability contributes to greatly enhanced Ca2+-sensitivity in skinned fibers. METHODS: Force of contraction in skinned cardiac porcine fiber and spectroscopic studies were performed. RESULTS: A decrease of -0.26 and -0.25 pCa units in Ca2+-sensitivity of contraction after PKA incubation was observed for skinned fibers incorporated with WT or cTnT-ΔE96, respectively. To further assess whether cTnT-ΔE96 interferes solely with transmission of cTnI phosphorylation effects, skinned fibers were reconstituted with PKA pseudo-phosphorylated cTnI (cTnI-SS/DD.cTnC). Fibers displaced with cTnT-WT, reconstituted with cTnI-SS/DD.cTnC decreased Ca2+-sensitivity of force (pCa50=5.61) compared to control cTnI-WT.cTnC (pCa50=5.75), similarly affecting cTnT-ΔE96 (pCa50=6.03) compared to control \cTnI-WT.cTnC (pCa50=6.14). Fluorescence studies measuring cTnC(IAANS) Ca2+-affinity changes due to cTnT-ΔE96 indicated that higher complexity (thin filament) better recapitulates skinned fiber Ca2+ sensitive changes. Circular dichroism revealed reduced α-helicity and earlier thermal unfolding for cTnT-ΔE96 compared to WT. CONCLUSIONS: Although ineffective in decreasing myofilament Ca2+-sensitivity to normal levels, cTnT-ΔE96 does not interfere with PKA cTnI phosphorylation mediated effects; 2) cTnT-ΔE96 requires actin to increase cTnC Ca2+-affinity; and 3) deletion of E96 reduces cTnT stability, likely disrupting crucial thin filament interactions. GENERAL SIGNIFICANCE: The pathological effect of cTnT-ΔE96 is largely manifested by dramatic myofilament Ca2+-sensitization which still persists even after PKA phosphorylation mediated Ca2+-desensitization.

Effect of Sealant Application and Thermal Cycling on Bond Strength of Tissue Conditioners to Acrylic Resin

The aim of this study was to evaluate the effect of sealer application and thermal cycling on the bond strength between tissue conditioners and acrylic resin, and to observe the type of bond failure. Two hundred eighty-eight specimens (10x16x3 mm) were made of an acrylic resin (Lucitone 500, Dentsply) using a metal muffle. Specimens were divided into four groups according to the tissue conditioner (Coe-Comfort, GC or Dentusoft, Densell) used and whether or not a sealer (Eversoft Soft Liner Sealer, Myerson) was applied. Each of the four groups was subdivided into other six subgroups (n=12) to undergo thermocycling for 45, 90, 135, 180 or 210 cycles with a dwell time of 60 s, or to be left non thermocycled (control). Tensile bond strength was measured in a universal testing machine at a crosshead speed of 5 mm/min. Sealant application had no effect on the tensile bond strength of the relined acrylic resin, regardless of the tissue conditioner used (Coe-Comfort: p=0.306 and Dentusoft: p=0.1501). The number of thermal cycles had a significant effect on the tensile bond strength of the relined acrylic resin (Coe-Comfort: p=0.002 and Dentusoft: p<0.001). Both tissue conditioners presented similar bond strength to acrylic resin. For both tissue conditioners, sealer coatings had no influence on bond strength, while different numbers of thermal cycles affected that mechanical property.

Os objetivos deste estudo foram avaliar o efeito da aplicação de selante e a ciclagem térmica na resistência de união entre condicionadores de tecido e resina acrílica e observar o padrão de fratura. Duzentos e oitenta e oito espécimes (10×16×3 mm) de resina acrílica (Lucitone 500, Dentsply) foram confeccionados utilizando-se uma mufla metálica. Os espécimens foram divididos em quatro grupos de acordo com o condicionador de tecido (Coe-Comfort, GC ou Dentusoft, Densell) e com o uso ou não de selante (Eversoft Soft Liner Sealer, Myerson). Cada um dos quatro grupos foi subdividido em seis grupos para a realização de 45, 90, 135, 180 ou 210 ciclos térmicos, ou ausência de termociclagem (controle). Resistência de união por tração foi mensurada em uma máquina universal de ensaios com velocidade de 5 mm/min. A aplicação de selante não afetou a resistência de união por tração da resina acrílica reembasada, independentemente do condicionador de tecido utilizado (Coe-Comfort: p=0,306 e Dentusoft: p=0,1501). O número de ciclos térmicos apresentou efeito significativo na resistência de união por tração da resina acrílica reembasada (Coe-Comfort: p=0,002 e Dentusoft: p<0,001). Ambos os condicionadores de tecidos apresentaram resistência de união semelhante à resina acrílica. Para ambos os condicionadores de tecidos, a aplicação de selante não apresentou influência na resistência de união, enquanto que diferentes números de ciclos térmicos afetaram essa propriedade mecânica.

The functional properties of human slow skeletal troponin T isoforms in cardiac muscle regulation.

Human slow skeletal troponin T (HSSTnT) shares a high degree of homology with cardiac TnT (CTnT). Although the presence of HSSTnT has not been confirmed in the heart at the protein level, detectable levels of HSSTnT mRNA have been found. Whether HSSTnT isoforms are expressed transiently remains unknown. Because transient re-expression of HSSTnT may be a potential mechanism of regulating function, we explored the effect of HSSTnT on the regulation of cardiac muscle. At least three HSSTnT isoforms have been found to exist in slow skeletal muscle: HSSTnT1 (+exons 5 and 12), HSSTnT2 (+exon 5, -exon 12), and HSSTnT3 (-exons 5 and 12). Another isoform, HSSTnT hypothetical (Hyp) (-exon 5, +exon 12), has only been found at the mRNA level. Compared with HCTnT3 (adult isoform), Tn complexes containing HSSTnT1, -2, and -3 did not alter the actomyosin ATPase activation and inhibition in the presence and absence of Ca(2+), respectively. HSSTnTHyp was not evaluated as it did not form a Tn complex under a variety of conditions. Porcine papillary skinned fibers displaced with HSSTnT1, -2, or -3 and reconstituted with human cardiac troponin I and troponin C (HCTnI·TnC) complex showed a decrease in the Ca(2+) sensitivity of force development and an increase in maximal recovered force (HSSTnT1 and -3) compared with HCTnT3. In contrast, HSSTnTHyp showed an increase in the Ca(2+) sensitivity of force development. This suggests that re- or overexpression of specific SSTnT isoforms might have therapeutic potential in the failing heart because they increase the maximal force of contraction. In addition, circular dichroism and proteolytic digestion experiments revealed structural differences between HSSTnT isoforms and HCTnT3 and that HSSTnT1 is more susceptible to calpain and trypsin proteolysis than the other HSSTnTs. Overall, HSSTnT isoforms despite being homologues of CTnT may display distinct functional properties in muscle regulation.

A mutation in TNNC1-encoded cardiac troponin C, TNNC1-A31S, predisposes to hypertrophic cardiomyopathy and ventricular fibrillation.

Defined as clinically unexplained hypertrophy of the left ventricle, hypertrophic cardiomyopathy (HCM) is traditionally understood as a disease of the cardiac sarcomere. Mutations in TNNC1-encoded cardiac troponin C (cTnC) are a relatively rare cause of HCM. Here, we report clinical and functional characterization of a novel TNNC1 mutation, A31S, identified in a pediatric HCM proband with multiple episodes of ventricular fibrillation and aborted sudden cardiac death. Diagnosed at age 5, the proband is family history-negative for HCM or sudden cardiac death, suggesting a de novo mutation. TnC-extracted cardiac skinned fibers were reconstituted with the cTnC-A31S mutant, which increased Ca(2+) sensitivity with no effect on the maximal contractile force generation. Reconstituted actomyosin ATPase assays with 50% cTnC-A31S:50% cTnC-WT demonstrated Ca(2+) sensitivity that was intermediate between 100% cTnC-A31S and 100% cTnC-WT, whereas the mutant increased the activation of the actomyosin ATPase without affecting the inhibitory qualities of the ATPase. The secondary structure of the cTnC mutant was evaluated by circular dichroism, which did not indicate global changes in structure. Fluorescence studies demonstrated increased Ca(2+) affinity in isolated cTnC, the troponin complex, thin filament, and to a lesser degree, thin filament with myosin subfragment 1. These results suggest that this mutation has a direct effect on the Ca(2+) sensitivity of the myofilament, which may alter Ca(2+) handling and contribute to the arrhythmogenesis observed in the proband. In summary, we report a novel mutation in the TNNC1 gene that is associated with HCM pathogenesis and may predispose to the pathogenesis of a fatal arrhythmogenic subtype of HCM.

Dimensional accuracy of impression material used in complete dentures / Fidelidade dimensional de materiais de moldagem funcional em prótese total

Objective: The aim of this research was to compare the degree of dimensional stability and accuracy of Sapphire (Bosworth, Skokle, Illinois, EUA), which is an ethyl methacrylate acrylic resin, with polyvinyl siloxane (ADSIL-Vigodent, Rio de Janeiro, Brazil), a very accurate material of worldwide use. Methods: The materials were tested by two techniques, the one-step putty and two-step putty-wash impression techniques. Stone casts were produced from impressions of an aluminum master cast of an edentulous maxilla. The samples were divided into four groups of 15 impressions each: Group 1 - putty-wash impression with Sapphire (putty) and Adsil Fluido (wash); Group 2 - putty impression with Sapphire; Group 3 - putty impression with Adsil Regular (putty); Group 4 - putty-wash impression with Adsil Regular (putty) and Adsil Fluido (wash). Measurements were done by the B251 Mitutoyo Coordinate Measuring Machine (Mitutoyo Co., Suzano, Brazil). The statistical treatment included calculating the mean, variance and standard deviation of the samples. The significance level was set at 5% (p<0.05). The distances found for each group were compared with those of the master cast by the t-test. Results: Sapphire putty impressions presented the best dimensional stability. On the other hand, polyvinyl siloxane putty (Adsil) presented significantdimensional variations in samples produced from putty and putty-wash impressions. When Sapphire (Bosworth, Skokie, Illinois, EUA) and Adsilwere used together (putty-wash), Sapphire (Bosworth, Skokie, Illinois, EUA) improved the results of the putty-wash impressions. Conclusion: In conclusion, Sapphire (Bosworth, Skokie, Illinois, EUA) is a reliable and stable material that can be used for complete denture impressions.

Objetivo: Comparar o nível de fidelidade e precisão do Sapphire (Bosworth, Skokle, Illinois, EUA), uma resina acrílica modificada, com um polivinilsiloxano (Adsil - Vigodent, Rio de Janeiro, Brasil), um material considerado de alta fidelidade e amplo uso. Métodos: Os materiais foram empregados em duas técnicas: moldagem simples e moldagem dupla. Os modelos em gesso foram construídos a partir de moldes obtidos do modelo padrão metálico de alumínio que reproduz uma maxila edêntula. Foram estabelecidos 4 grupos de 15 moldagens assim divididos: Grupo 1 - Sapphire reembasado com Adsil Fluido; Grupo 2 - Sapphire com camada única; Grupo 3 - Adsil Regular com camada única e Grupo 4 - Adsil Regular mais Adsil Fluido com camada dupla. As medidas foram realizadas por um aparelho de medição por coordenadas B251(Mitutoyo Co., Suzano, Brasil). Na análise estatística foram calculados a média, variância e o desvio padrão das amostras (nível de significância<0,05). Em seguida foi feita uma comparação da distancia encontrada em cada grupo com o valor padrão através do teste t para uma amostra.Resultados: O Sapphire (Bosworth, Skokie, Illinois, EUA), em moldagem simples, apresentou melhores resultados em relação à estabilidade dimensional. Ao contrário do silicone de adição (Adsil, Rio de Janeiro, Brasil) que apresentou variações dimensionais significativas nos modelos obtidos por moldagem simples e também em moldagem dupla. O Sapphire (Bosworth, Skokie, Illinois, EUA) melhorou os resultados do silicone de adição ao ser misturado com este em moldagem dupla. Conclusão: Pode-se concluir que o Sapphire (Bosworth, Skokie, Illinois, EUA) é um material de moldagem fiel e de uso viável para moldagens em PróteseTotal.

Generation and functional characterization of knock-in mice harboring the cardiac troponin I-R21C mutation associated with hypertrophic cardiomyopathy.

The R21C substitution in cardiac troponin I (cTnI) is the only identified mutation within its unique N-terminal extension that is associated with hypertrophic cardiomyopathy (HCM) in man. Particularly, this mutation is located in the consensus sequence for ß-adrenergic-activated protein kinase A (PKA)-mediated phosphorylation. The mechanisms by which this mutation leads to heart disease are still unclear. Therefore, we generated cTnI knock-in mouse models carrying an R21C mutation to evaluate the resultant functional consequences. Measuring the in vivo levels of incorporated mutant and WT cTnI, and their basal phosphorylation levels by top-down mass spectrometry demonstrated: 1) a dominant-negative effect such that, the R21C+/- hearts incorporated 24.9% of the mutant cTnI within the myofilament; and 2) the R21C mutation abolished the in vivo phosphorylation of Ser(23)/Ser(24) in the mutant cTnI. Adult heterozygous (R21C+/-) and homozygous (R21C+/+) mutant mice activated the fetal gene program and developed a remarkable degree of cardiac hypertrophy and fibrosis. Investigation of cardiac skinned fibers isolated from WT and heterozygous mice revealed that the WT cTnI was completely phosphorylated at Ser(23)/Ser(24) unless the mice were pre-treated with propranolol. After propranolol treatment (-PKA), the pCa-tension relationships of all three mice (i.e. WT, R21C+/-, and R21C+/+) were essentially the same. However, after treatment with propranolol and PKA, the R21C cTnI mutation reduced (R21C+/-) or abolished (R21C+/+) the well known decrease in the Ca(2+) sensitivity of tension that accompanies Ser(23)/Ser(24) cTnI phosphorylation. Altogether, the combined effects of the R21C mutation appear to contribute toward the development of HCM and suggest that another physiological role for the phosphorylation of Ser(23)/Ser(24) in cTnI is to prevent cardiac hypertrophy.

Avaliação da influência de diferentes técnicas de moldagem na desadaptação de infraestrutura em implantes múltiplos angulados / Evaluation of the influence of different impression techniques on misfit of superstructure in multiple angled implants

Neste estudo avaliou-se a desadaptação de uma infraestrutura, utilizando três técnicas de moldagem de transferência, divididas em três grupos: (G1) transferentes cônicos; (G2) transferentes quadrados sem união; (G3) transferentes quadrados unidos com fio dental e resina acrílica. Foram confeccionados cinco corpos de prova para cada grupo (n = 5). Todas as moldagens foram realizadas com poliéter (Impregum, 3M Espe). Confeccionou-se um modelo mestre em acrílico a partir do modelo de uma mandíbula edêntula com quatro implantes, sendo os dois centrais paralelos e os dois das extremidades inclinados próximos aos 15° para distal. Confeccionou-se, sobre estes, uma infraestrutura mestre, que serviu como situação controle (C). A avaliação das desadaptações foi feita por meio da técnica do parafuso único e as mensurações foram realizadas utilizando um microscópio de medição universal nos corpos de prova, todos pela face vestibular. Para identificar a angulação dos implantes distais no modelo mestre, foi utilizada uma máquina de medição por coordenadas. Os dados foram analisados em micrômetros, adotando nível de significância 5%. Os resultados demonstraram que o grupo G1 apresentou média significativamente maior entre os grupos (p = 359,2 µm), o grupo G2 não se diferenciou de forma significante dos demais grupos (p = 274,6 µm) e o grupo G3 apresentou a menor média (p = 116,3 µm). Pôde-se concluir que o grupo G3, que utilizou transferentes quadrados unidos, apresentou os melhores resultados entre as técnicas avaliadas neste estudo

In this study the misfit of a substructure was evaluated by using three impression transfer techniques, divided into three groups: (G1) tapered transfers; (G2) non-splinted square transfers, and (G3) square transfers splinted together with dental floss/precision acrylic resin. Five test specimens were fabricated per group (n = 5). All impressions were taken with polyether material (Impregum, 3M Espe). An acrylic master model was made with four implants: two parallel central implants and two distal implants angled at 15°. After, a master substructure was fabricated and used as control (C). Misfits were evaluated by means of the one-screw test protocol, and all the measurements of the test specimens were taken on the buccal aspect, using a Universal Measuring Microscope. A coordinate measuring machine (CMM) was used to determine the distal implant angulations in the master model. Data were analyzed using the Anova test (alfa = 5%). The results demonstrated that Group G1 presented a significantly higher mean than other groups (p = 359.2micrometers). The group G2 did not differ significantly from the other groups (p = 274.6 micrometers), and group G3 presented the lowest mean (p =116.3 micrometers). It can be concluded that Group G3 presented the best results among the techniques evaluated in this study.

Análise da utilização da união dente/implante e tipos de conexões: revisão de literatura / Analysis of the use of the tooth-implant union and types of connections: literature review

Após a divulgação do conceito de osseointegração por Brãnemark, baseado em anos de estudo, houve uma grande mudança na Odontologia moderna. Os tratamentos ganharam muitas variáveis no planejamento, normalmente mais estéticos e eficazes. Apesar da introdução dos implantes osseointegrados, com grande demanda e evolução da técnica, alguns fatores ainda limitam a colocação de um número adequado de implantes. Com isso, tem-se deparado com uma situação clínica de grande importância na Implantodontia: a restauração do edentulismo parcial utilizando pilares de dentes naturais unidos a implantes. O principal fator de preocupação ao se indicar a união dente/implante é a biomecânica, devido à diferença de absorção das forças oclusais pelos dois componentes distintos, dente e implante. Através de uma revisão de literatura, foram abordados os critérios biomecânicos dessa união, considerações sobre as formas de executá-la, as possíveis complicações e os achados clínicos das pesquisas. Embora os conceitos teóricos revelem problemas em potencial, os resultados clínicos apontam favoravelmente para o uso desse tipo de união. Segundo alguns profissionais, principalmente em segmentos curtos e com conexões rígidas, entretanto, o assunto é ainda bastante controverso.

After the popularization of the osseointegration concept from Branemark, based on years of study, there was a great turn in the modem dentistry. The treatments won many variation during prosthodontic planning related to aesthetics and efficacy. In spite of introduction of osseointegrated implants, technical developments and increase of indications, several factors still limit the use of an appropriate number of implants. With that, a critical situation in the Implantology: to use or not tooth-implant connection, and in using, what is the best way to do so. The main factor of relevance to indicate the tooth-implant connection is the biomechanics, due to the difference of absorption of the occlusal forces on the two different components, tooth and implant. Through literature review, the biomechanical criteria of this union, considerations of how to do it possible and the complications that could occur were discussed. Although the theoretical concepts reveal potentials problems, the clinical results appear favorable to the use of this type of union, mainly in short segments and with rigid connections.

Redox state of troponin C cysteine in the D/E helix alters the C-domain affinity for the thin filament of vertebrate striated muscle.

BACKGROUND: Despite a broad spectrum of structural studies, it is not yet clear whether the D/E helix of troponin C (TnC) contributes to the interaction of TnC with troponin I (TnI). Redox modifications at Cys 98 in the D/E helix were explored for clues to TnC binding to the thin filament off-state, using recombinant wild-type TnC and an engineered mutant without Cys (Cys98Leu). METHODS: Recombinant proteins and rabbit psoas skinned fibres were reduced with dithiothreitol (DTT) and variously recombined. Changes in affinity of reduced or oxidised TnC for the thin filament were evaluated via TnC binding and dissociation, using a standardized test for maximal force as an index of fibre TnC content. RESULTS: All oxidation and reduction effects observed were reversible and led to changes in TnC content. Oxidation (H(2)O(2)) reduced TnC affinity for the filament; reduction (DTT) increased it. Reducing other fibre proteins had no effect. Binding of the Cys-less TnC mutant was not altered by DTT, nor was dissociation of wild-type TnC from reconstituted hybrids (skeletal TnC in cardiac trabeculae). Thus when Cys 98 in the D/E helix of TnC is fully reduced, its binding affinity for the thin filament of skeletal muscle is enhanced and helps to anchor it to the filament. GENERAL SIGNIFICANCE: Signal transmission between TnC and the other proteins of the regulatory complex is sensitive to the redox state of Cys 98.

Avaliação da adaptação entre componentes protéticos e implantes / Evaluation of marginal adaptation between dental implants and prosthetic components

O objetivo deste trabalho foi avaliar, por meio de um microdurômetro, possíveis diferenças na adaptação de diferentes componentes protéticos quando colocados em uma mesma marca comercial de implantes. Foram utilizados 30 implantes de hexágono externo da marca comercial Neodent e 30 componentes protéticos tipo pilar Ucla de titânio com hexágono externo, plataforma regular de três diferentes marcas comerciais: Neodent, Conexão e Titaniumfix (dez de cada). Cada implante recebeu apenas um componente, que foi anexado ao implante por meio de um parafuso de titânio, utilizando-se torque de 20 Ncm, com torquímetro manual. Cada conjunto implante/componente recebeu quatro marcações aleatórias em torno da circunferência, onde foram feitas as medições do espaço existente entre as peças, usando-se uma lente de 200 vezes de aumento. Os resultados obtidos não mostraram diferenças estatisticamente significantes entre os grupos, submetidos ao teste Anova (p = 0,05). Concluiu-se que há compatibilidade entre os componentes das três diferentes marcas utilizadas com os implantes da Neodent.

The aim of this study was to evaluate possible differences on margin fit of prosthetic components from several dental implant companies. Thirty external hexagon type implants (Neodent) received 30 prosthetic Ucla-type components from three different companies: Conexão (n = 10), Neodent (n = 10), and Titaniumfix (n = 10). All componentes were tightened to 20 Ncm using a torque driver. Each set (implant/component), received four random demarcations around the circumference, where gap measurement was performed under 200 X using a microhardness indenter. No statistically significant differences were observed among groups (Anova test, p > .05). It can be concluded that all prosthetic component are compatible with external hex Neodent implants.