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1.
Food Chem ; 372: 131347, 2022 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-34818749

RESUMO

The subcellular distribution of calpain-1 and -2 and the proteolytical activity of myofibril-bound calpains in pork were investigated during 12 days cold storage. The content of sarcoplasmic calpain-1 decreased during storage while myofibril-bound calpain-1 content first increased (P < 0.05) to 17% of that of 12 h-sarcoplasmic calpain-1 on day 6 followed by a gradual decrease with subsequent storage, suggesting that calpain-1 gradually translocated from sarcoplasm to myofibrils during the initial 6 days of postmortem storage. Intact desmin decreased (P < 0.05) after incubation of myofibrils with 0.05 mM Ca2+, and this was more pronounced with 5 mM Ca2+ (P < 0.05). Ca2+ titration curves of day 6 myofibrils showed two distinct proteolytic activities becoming activated in the range 0.03 to 0.06 mM and 0.4 to 0.8 mM Ca2+, respectively. The results suggest that both calpain-1 and calpain-2 binds to myofibrils during storage and subsequently degrade structural proteins including desmin.


Assuntos
Carne de Porco , Carne Vermelha , Animais , Calpaína/genética , Calpaína/metabolismo , Desmina/genética , Desmina/metabolismo , Carne/análise , Músculo Esquelético/metabolismo , Miofibrilas/metabolismo , Mudanças Depois da Morte , Proteólise , Suínos
2.
Cells ; 10(10)2021 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-34685769

RESUMO

Life-threatening ventricular arrhythmias are the main clinical burden in patients with hypertrophic cardiomyopathy (HCM), and frequently occur in young patients with mild structural disease. While massive hypertrophy, fibrosis and microvascular ischemia are the main mechanisms underlying sustained reentry-based ventricular arrhythmias in advanced HCM, cardiomyocyte-based functional arrhythmogenic mechanisms are likely prevalent at earlier stages of the disease. In this review, we will describe studies conducted in human surgical samples from HCM patients, transgenic animal models and human cultured cell lines derived from induced pluripotent stem cells. Current pieces of evidence concur to attribute the increased risk of ventricular arrhythmias in early HCM to different cellular mechanisms. The increase of late sodium current and L-type calcium current is an early observation in HCM, which follows post-translation channel modifications and increases the occurrence of early and delayed afterdepolarizations. Increased myofilament Ca2+ sensitivity, commonly observed in HCM, may promote afterdepolarizations and reentry arrhythmias with direct mechanisms. Decrease of K+-currents due to transcriptional regulation occurs in the advanced disease and contributes to reducing the repolarization-reserve and increasing the early afterdepolarizations (EADs). The presented evidence supports the idea that patients with early-stage HCM should be considered and managed as subjects with an acquired channelopathy rather than with a structural cardiac disease.


Assuntos
Arritmias Cardíacas/complicações , Arritmias Cardíacas/metabolismo , Cálcio/metabolismo , Cardiomiopatia Hipertrófica/complicações , Cardiomiopatia Hipertrófica/metabolismo , Canais Iônicos/metabolismo , Miofibrilas/metabolismo , Animais , Arritmias Cardíacas/fisiopatologia , Cardiomiopatia Hipertrófica/fisiopatologia , Humanos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia
3.
Int J Mol Sci ; 22(17)2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34502534

RESUMO

Rare pediatric non-compaction and restrictive cardiomyopathy are usually associated with a rapid and severe disease progression. While the non-compaction phenotype is characterized by structural defects and is correlated with systolic dysfunction, the restrictive phenotype exhibits diastolic dysfunction. The molecular mechanisms are poorly understood. Target genes encode among others, the cardiac troponin subunits forming the main regulatory protein complex of the thin filament for muscle contraction. Here, we compare the molecular effects of two infantile de novo point mutations in TNNC1 (p.cTnC-G34S) and TNNI3 (p.cTnI-D127Y) leading to severe non-compaction and restrictive phenotypes, respectively. We used skinned cardiomyocytes, skinned fibers, and reconstituted thin filaments to measure the impact of the mutations on contractile function. We investigated the interaction of these troponin variants with actin and their inter-subunit interactions, as well as the structural integrity of reconstituted thin filaments. Both mutations exhibited similar functional and structural impairments, though the patients developed different phenotypes. Furthermore, the protein quality control system was affected, as shown for TnC-G34S using patient's myocardial tissue samples. The two troponin targeting agents levosimendan and green tea extract (-)-epigallocatechin-3-gallate (EGCg) stabilized the structural integrity of reconstituted thin filaments and ameliorated contractile function in vitro in some, but not all, aspects to a similar degree for both mutations.


Assuntos
Cardiomiopatias/genética , Mutação de Sentido Incorreto , Miofibrilas/metabolismo , Troponina I/genética , Adenosina Trifosfatases/metabolismo , Adulto , Cálcio/metabolismo , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Catequina/análogos & derivados , Catequina/farmacologia , Humanos , Lactente , Masculino , Microscopia Eletrônica de Transmissão , Miofibrilas/efeitos dos fármacos , Miofibrilas/ultraestrutura , Sarcômeros/efeitos dos fármacos , Sarcômeros/metabolismo , Índice de Gravidade de Doença , Simendana/farmacologia , Tropomiosina/metabolismo , Troponina I/metabolismo
4.
Methods Mol Biol ; 2319: 15-24, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331238

RESUMO

Mouse models are extensively studied and well-used to study cardiomyopathies due to the genetic relevance of this model organism and increasing need to identify therapeutic targets. Cardiac myofibril preparation for whole hearts has proved to be an invaluable in vitro method for determining the fundamental molecular mechanisms in heart failure. The technique described below consistently yields intact cardiac myofibrils, which can be used in subsequent techniques such as western blotting, immunofluorescence, and mass spectrometry. Here, we describe a method to optimize the separation and yield of cardiac myofibrils from murine whole tissue samples and preparation for subsequent mass spectrometry. This method allows for quick visual identification of multiple cardiac myofibril proteins.


Assuntos
Espectrometria de Massas/métodos , Miocárdio/metabolismo , Miofibrilas/metabolismo , Animais , Coração , Camundongos
5.
Int J Mol Sci ; 22(12)2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-34204776

RESUMO

Point mutations in the genes encoding the skeletal muscle isoforms of tropomyosin can cause a range of muscle diseases. The amino acid substitution of Arg for Pro residue in the 90th position (R90P) in γ-tropomyosin (Tpm3.12) is associated with congenital fiber type disproportion and muscle weakness. The molecular mechanisms underlying muscle dysfunction in this disease remain unclear. Here, we observed that this mutation causes an abnormally high Ca2+-sensitivity of myofilaments in vitro and in muscle fibers. To determine the critical conformational changes that myosin, actin, and tropomyosin undergo during the ATPase cycle and the alterations in these changes caused by R90P replacement in Tpm3.12, we used polarized fluorimetry. It was shown that the R90P mutation inhibits the ability of tropomyosin to shift towards the outer domains of actin, which is accompanied by the almost complete depression of troponin's ability to switch actin monomers off and to reduce the amount of the myosin heads weakly bound to F-actin at a low Ca2+. These changes in the behavior of tropomyosin and the troponin-tropomyosin complex, as well as in the balance of strongly and weakly bound myosin heads in the ATPase cycle may underlie the occurrence of both abnormally high Ca2+-sensitivity and muscle weakness. BDM, an inhibitor of myosin ATPase activity, and W7, a troponin C antagonist, restore the ability of tropomyosin for Ca2+-dependent movement and the ability of the troponin-tropomyosin complex to switch actin monomers off, demonstrating a weakening of the damaging effect of the R90P mutation on muscle contractility.


Assuntos
Contração Muscular/genética , Mutação/genética , Oximas/farmacologia , Sulfonamidas/farmacologia , Tropomiosina/genética , Actinas/metabolismo , Animais , Cálcio/metabolismo , Contração Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Miofibrilas/efeitos dos fármacos , Miofibrilas/metabolismo , Miosinas/metabolismo , Coelhos , Troponina/metabolismo
6.
Nat Commun ; 12(1): 3715, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140474

RESUMO

A comprehensive transcriptomic survey of pigs can provide a mechanistic understanding of tissue specialization processes underlying economically valuable traits and accelerate their use as a biomedical model. Here we characterize four transcript types (lncRNAs, TUCPs, miRNAs, and circRNAs) and protein-coding genes in 31 adult pig tissues and two cell lines. We uncover the transcriptomic variability among 47 skeletal muscles, and six adipose depots linked to their different origins, metabolism, cell composition, physical activity, and mitochondrial pathways. We perform comparative analysis of the transcriptomes of seven tissues from pigs and nine other vertebrates to reveal that evolutionary divergence in transcription potentially contributes to lineage-specific biology. Long-range promoter-enhancer interaction analysis in subcutaneous adipose tissues across species suggests evolutionarily stable transcription patterns likely attributable to redundant enhancers buffering gene expression patterns against perturbations, thereby conferring robustness during speciation. This study can facilitate adoption of the pig as a biomedical model for human biology and disease and uncovers the molecular bases of valuable traits.


Assuntos
Tecido Adiposo/metabolismo , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , RNA Circular/metabolismo , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Transcriptoma/genética , Processamento Alternativo , Animais , Evolução Biológica , Linhagem Celular , Linhagem da Célula , Núcleo Celular/genética , Núcleo Celular/metabolismo , Elementos Facilitadores Genéticos , Evolução Molecular , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , MicroRNAs/genética , Mitocôndrias/metabolismo , Conformação Molecular , Miofibrilas/genética , Miofibrilas/metabolismo , Filogenia , Regiões Promotoras Genéticas , RNA Circular/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Análise Espacial , Suínos
7.
Methods Mol Biol ; 2277: 405-421, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080165

RESUMO

The more recent studies of human pathologies have essentially revealed the complexity of the interactions involved at the different levels of integration in organ physiology. Integrated organ thus reveals functional properties not predictable by underlying molecular events. It is therefore obvious that current fine molecular analyses of pathologies should be fruitfully combined with integrative approaches of whole organ function. It follows that an important issue in the comprehension of the link between molecular events in pathologies and whole organ function/dysfunction is the development of new experimental strategies aimed at the study of the integrated organ physiology. Cardiovascular diseases are a good example as heart submitted to ischemic conditions has to cope both with a decreased supply of nutrients and oxygen, and the necessary increased activity required to sustain whole body-including the heart itself-oxygenation.By combining the principles of control analysis with noninvasive 31P NMR measurement of the energetic intermediates and simultaneous measurement of heart contractile activity, we developed MoCA (for Modular Control and regulation Analysis), an integrative approach designed to study in situ control and regulation of cardiac energetics during contraction in intact beating perfused isolated heart (Diolez et al., Am J Physiol Regul Integr Comp Physiol 293(1):R13-R19, 2007). Because it gives real access to integrated organ function, MoCA brings out a new type of information-the "elasticities," referring to integrated internal responses to metabolic changes-that may be a key to the understanding of the processes involved in pathologies. MoCA can potentially be used not only to detect the origin of the defects associated with the pathology, but also to provide the quantitative description of the routes by which these defects-or also drugs-modulate global heart function, therefore opening therapeutic perspectives. This review presents selected examples of the applications to isolated intact beating heart that evidence different modes of energetic regulation of cardiac contraction. We also discuss the clinical application by using noninvasive 31P cardiac energetics examination under clinical conditions for detection of heart pathologies.


Assuntos
Metabolismo Energético , Espectroscopia de Ressonância Magnética/métodos , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Animais , Cálcio/metabolismo , Cardiotônicos/farmacologia , Metabolismo Energético/efeitos dos fármacos , Epinefrina/metabolismo , Cobaias , Coração/efeitos dos fármacos , Homeostase , Humanos , Masculino , Mitocôndrias Cardíacas/metabolismo , Miofibrilas/metabolismo , Técnicas de Cultura de Órgãos/métodos , Ratos , Simendana/farmacologia
8.
Nat Commun ; 12(1): 3852, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158500

RESUMO

Vertebrate muscles and tendons are derived from distinct embryonic origins yet they must interact in order to facilitate muscle contraction and body movements. How robust muscle tendon junctions (MTJs) form to be able to withstand contraction forces is still not understood. Using techniques at a single cell resolution we reexamine the classical view of distinct identities for the tissues composing the musculoskeletal system. We identify fibroblasts that have switched on a myogenic program and demonstrate these dual identity cells fuse into the developing muscle fibers along the MTJs facilitating the introduction of fibroblast-specific transcripts into the elongating myofibers. We suggest this mechanism resulting in a hybrid muscle fiber, primarily along the fiber tips, enables a smooth transition from muscle fiber characteristics towards tendon features essential for forming robust MTJs. We propose that dual characteristics of junctional cells could be a common mechanism for generating stable interactions between tissues throughout the musculoskeletal system.


Assuntos
Fibroblastos/metabolismo , Junções Intercelulares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Miofibrilas/metabolismo , Tendões/metabolismo , Animais , Fusão Celular , Células Cultivadas , Fibroblastos/citologia , Expressão Gênica , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Contração Muscular/genética , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/citologia , Sistema Musculoesquelético/citologia , Sistema Musculoesquelético/metabolismo , RNA-Seq/métodos , Tendões/citologia
9.
Proc Natl Acad Sci U S A ; 118(23)2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34088833

RESUMO

Changes in the molecular properties of cardiac myosin strongly affect the interactions of myosin with actin that result in cardiac contraction and relaxation. However, it remains unclear how myosin molecules work together in cardiac myofilaments and which properties of the individual myosin molecules impact force production to drive cardiac contractility. Here, we measured the force production of cardiac myofilaments using optical tweezers. The measurements revealed that stepwise force generation was associated with a higher frequency of backward steps at lower loads and higher stall forces than those of fast skeletal myofilaments. To understand these unique collective behaviors of cardiac myosin, the dynamic responses of single cardiac and fast skeletal myosin molecules, interacting with actin filaments, were evaluated under load. The cardiac myosin molecules switched among three distinct conformational positions, ranging from pre- to post-power stroke positions, in 1 mM ADP and 0 to 10 mM phosphate solution. In contrast to cardiac myosin, fast skeletal myosin stayed primarily in the post-power stroke position, suggesting that cardiac myosin executes the reverse stroke more frequently than fast skeletal myosin. To elucidate how the reverse stroke affects the force production of myofilaments and possibly heart function, a simulation model was developed that combines the results from the single-molecule and myofilament experiments. The results of this model suggest that the reversal of the cardiac myosin power stroke may be key to characterizing the force output of cardiac myosin ensembles and possibly to facilitating heart contractions.


Assuntos
Contração Miocárdica , Miocárdio/metabolismo , Miofibrilas/metabolismo , Miosinas/metabolismo , Animais , Suínos
10.
Am J Physiol Cell Physiol ; 321(1): C94-C103, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33979211

RESUMO

Cellular senescence is the irreversible arrest of normally dividing cells and is driven by cell cycle inhibitory proteins such as p16, p21, and p53. When cells enter senescence, they secrete a host of proinflammatory factors known as the senescence-associated secretory phenotype, which has deleterious effects on surrounding cells and tissues. Little is known of the role of senescence in Duchenne muscular dystrophy (DMD), the fatal X-linked neuromuscular disorder typified by chronic inflammation, extracellular matrix remodeling, and a progressive loss in muscle mass and function. Here, we demonstrate using C57-mdx (8-wk-old) and D2-mdx (4-wk-old and 8-wk-old) mice, two mouse models of DMD, that cells displaying canonical markers of senescence are found within the skeletal muscle. Eight-week-old D2-mdx mice, which display severe muscle pathology, had greater numbers of senescent cells associated with areas of inflammation, which were mostly Cdkn1a-positive macrophages, whereas in C57-mdx muscle, senescent populations were endothelial cells and macrophages localized to newly regenerated myofibers. Interestingly, this pattern was similar to cardiotoxin (CTX)-injured wild-type (WT) muscle, which experienced a transient senescent response. Dystrophic muscle demonstrated significant upregulations in senescence pathway genes [Cdkn1a (p21), Cdkn2a (p16INK4A), and Trp53 (p53)], which correlated with the quantity of senescence-associated ß-galactosidase (SA-ß-Gal)-positive cells. These results highlight an underexplored role for cellular senescence in murine dystrophic muscle.


Assuntos
Senescência Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Células Endoteliais/metabolismo , Macrófagos/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/genética , Animais , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Modelos Animais de Doenças , Distrofina/deficiência , Distrofina/genética , Células Endoteliais/patologia , Regulação da Expressão Gênica , Humanos , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/patologia , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Miofibrilas/metabolismo , Miofibrilas/patologia , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
11.
Am J Physiol Heart Circ Physiol ; 320(6): H2339-H2350, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33989081

RESUMO

Mutations to the sarcomere-localized cochaperone protein Bcl2-associated athanogene 3 (BAG3) are associated with dilated cardiomyopathy (DCM) and display greater penetrance in male patients. Decreased protein expression of BAG3 is also associated with nongenetic heart failure; however, the factors regulating cardiac BAG3 expression are unknown. Using left ventricular (LV) tissue from nonfailing and DCM human samples, we found that whole LV BAG3 expression was not significantly impacted by DCM or sex; however, myofilament localized BAG3 was significantly decreased in males with DCM. Females with DCM displayed no changes in BAG3 compared with nonfailing. This sex difference appears to be estrogen independent, as estrogen treatment in ovariectomized female rats had no impact on BAG3 expression. BAG3 gene expression in noncardiac cells is primarily regulated by the heat shock transcription factor-1 (HSF-1). We show whole LV HSF-1 expression and nuclear localized/active HSF-1 each displayed a striking positive correlation with whole LV BAG3 expression. We further found that HSF-1 localizes to the sarcomere Z-disc in cardiomyocytes and that this myofilament-associated HSF-1 pool decreases in heart failure. The decrease of HSF-1 was more pronounced in male patients and tightly correlated with myofilament BAG3 expression. Together our findings indicate that cardiac BAG3 expression and myofilament localization are differentially impacted by sex and disease and are linked to HSF-1.NEW & NOTEWORTHY Myofilament BAG3 expression decreases in male patients with nonischemic DCM but is preserved in female patients with DCM. BAG3 expression in the human heart is tightly linked to HSF-1 expression and nuclear translocation. HSF-1 localizes to the sarcomere Z-disc in the human heart. HSF-1 expression in the myofilament fraction decreases in male patients with DCM and positively correlates with myofilament BAG3.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Cardiomiopatia Dilatada/metabolismo , Ventrículos do Coração/metabolismo , Fatores de Transcrição de Choque Térmico/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Sarcômeros/metabolismo , Adulto , Idoso , Animais , Feminino , Expressão Gênica , Humanos , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Miocárdio/patologia , Miócitos Cardíacos/patologia , Miofibrilas/metabolismo , Ovariectomia , Ratos , Sarcômeros/patologia , Fatores Sexuais
12.
J Food Sci ; 86(6): 2387-2397, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34018189

RESUMO

Gelation properties of myofibrillar protein (MP)/wheat gluten (WG) induced by glutamine transaminase (TGase) were studied. Results showed that the inclusion of transglutaminase increased the gel strength, water-holding capacity (WHC), and nonfreezable water (Wnf) of MP/WG mixture. Circular dichroism (CD) analysis showed that the ß-sheet and random coil content of the MP/WG treated with TGase addition increased by 12.1% and 3.7%, while the α-helix and ß-turn content decreased by 14.2% and 1.8%. Rheological measurements showed that TGase induced higher energy storage modulus value during the MP/WG gel heating-cooling cycle. the hydrogen bond and hydrophobic interaction content of the MP/WG gels increased by 80 and 120 ug/L, and the disulfide bond decreased by 200 ug/L, with TGase addition was increased from 0 to 120 U/g protein. Scanning electron microscope (SEM) showed that MP/WG gel with TGase had uniform and dense network structure. PRACTICAL APPLICATION: The properties of myofibrillar/wheat gluten gel induced by TGase crosslinking was studied. The gel structure and water holding capacity of MP/WG were improved by the cross-linking of TGase. The study of the gel properties of MP/WG induced by TGase crosslinking also can provide a theoretical basis for analyzing the effect of TGase on the application of gluten protein in complex meat emulsion system.


Assuntos
Géis/química , Glutens/química , Miofibrilas/metabolismo , Reologia , Transglutaminases/farmacologia , Triticum/química , Glutens/efeitos dos fármacos , Glutens/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Miofibrilas/efeitos dos fármacos , Triticum/efeitos dos fármacos , Triticum/metabolismo
13.
Nat Commun ; 12(1): 3094, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34035273

RESUMO

Short-term, systemic expression of the Yamanaka reprogramming factors (Oct-3/4, Sox2, Klf4 and c-Myc [OSKM]) has been shown to rejuvenate aging cells and promote tissue regeneration in vivo. However, the mechanisms by which OSKM promotes tissue regeneration are unknown. In this work, we focus on a specific tissue and demonstrate that local expression of OSKM, specifically in myofibers, induces the activation of muscle stem cells or satellite cells (SCs), which accelerates muscle regeneration in young mice. In contrast, expressing OSKM directly in SCs does not improve muscle regeneration. Mechanistically, expressing OSKM in myofibers regulates the expression of genes important for the SC microenvironment, including upregulation of p21, which in turn downregulates Wnt4. This is critical because Wnt4 is secreted by myofibers to maintain SC quiescence. Thus, short-term induction of the Yamanaka factors in myofibers may promote tissue regeneration by modifying the stem cell niche.


Assuntos
Diferenciação Celular/genética , Reprogramação Celular/genética , Miofibrilas/metabolismo , Regeneração/genética , Células Satélites de Músculo Esquelético/metabolismo , Nicho de Células-Tronco , Animais , Células Cultivadas , Feminino , Expressão Gênica , Fatores de Transcrição Kruppel-Like/genética , Camundongos Transgênicos , Miofibrilas/fisiologia , Fator 3 de Transcrição de Octâmero/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/genética , Células Satélites de Músculo Esquelético/citologia , Proteína Wnt4/genética
14.
Food Chem ; 361: 130100, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34044215

RESUMO

The purpose of this study was to investigate the structural properties of µ-calpain induced by hydroxyl radical oxidation and its effect on the degradation of myofibrillar protein (MP) from the dorsal muscles of Coregonus peled. The carbonyl and sulfhydryl content of µ-calpain changed significantly after oxidation. The content of α-helix in the secondary structure decreased from 0.825 to 0.232 and the changes in intrinsic fluorescence and ultraviolet (UV) absorption spectra indicated that oxidation could cause the expansion and aggregation of µ-calpain molecules. Changes in µ-calpain structure could improve the activity of µ-calpain, reaching the highest value at 0.5 mM H2O2. The highest µ-calpain activity facilitate the degradation of unoxidized MP, while the degradation of oxidized MP was facilitated at the 1 mM H2O2. Thus, our results provide a scientific basis for the interaction mechanism among hydroxyl radical oxidation, µ-calpain, and MP degradation.


Assuntos
Calpaína/metabolismo , Proteínas Musculares/metabolismo , Miofibrilas/metabolismo , Proteólise , Salmonidae/metabolismo , Animais , Calpaína/química , Peróxido de Hidrogênio/metabolismo , Oxirredução
15.
Nat Commun ; 12(1): 2091, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33828099

RESUMO

Complex animals build specialised muscles to match specific biomechanical and energetic needs. Hence, composition and architecture of sarcomeres and mitochondria are muscle type specific. However, mechanisms coordinating mitochondria with sarcomere morphogenesis are elusive. Here we use Drosophila muscles to demonstrate that myofibril and mitochondria morphogenesis are intimately linked. In flight muscles, the muscle selector spalt instructs mitochondria to intercalate between myofibrils, which in turn mechanically constrain mitochondria into elongated shapes. Conversely in cross-striated leg muscles, mitochondria networks surround myofibril bundles, contacting myofibrils only with thin extensions. To investigate the mechanism causing these differences, we manipulated mitochondrial dynamics and found that increased mitochondrial fusion during myofibril assembly prevents mitochondrial intercalation in flight muscles. Strikingly, this causes the expression of cross-striated muscle specific sarcomeric proteins. Consequently, flight muscle myofibrils convert towards a partially cross-striated architecture. Together, these data suggest a biomechanical feedback mechanism downstream of spalt synchronizing mitochondria with myofibril morphogenesis.


Assuntos
Mitocôndrias/metabolismo , Morfogênese/fisiologia , Músculo Esquelético/metabolismo , Miofibrilas/metabolismo , Animais , Fenômenos Biomecânicos , Drosophila , Proteínas de Drosophila , Drosophila melanogaster , Retroalimentação , Voo Animal/fisiologia , Masculino , Fenômenos Mecânicos , Mitocôndrias/ultraestrutura , Desenvolvimento Muscular , Músculo Esquelético/citologia , Miofibrilas/ultraestrutura , Fatores de Regulação Miogênica , Sarcômeros/metabolismo , Fatores de Transcrição
16.
J Biol Chem ; 296: 100228, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33814345

RESUMO

The conserved C-terminal end segment of troponin I (TnI) plays a critical role in regulating muscle relaxation. This function is retained in the isolated C-terminal 27 amino acid peptide (residues 184-210) of human cardiac TnI (HcTnI-C27): When added to skinned muscle fibers, HcTnI-C27 reduces the Ca2+-sensitivity of activated myofibrils and facilitates relaxation without decreasing the maximum force production. However, the underlying mechanism of HcTnI-C27 function is unknown. We studied the conformational preferences of HcTnI-C27 and a myopathic mutant, Arg192His, (HcTnI-C27-H). Both peptides were mainly disordered in aqueous solution with a nascent helix involving residues from Trp191 to Ile195, as shown by NMR analysis and molecular dynamics simulations. The population of nascent helix was smaller in HcTnI-C27-H than in HcTnI-C27, as shown by circular dichroism (CD) titrations. Fluorescence and isothermal titration calorimetry (ITC) showed that both peptides bound tropomyosin (αTm), with a detectably higher affinity (∼10 µM) of HcTnI-C27 than that of HcTnI-C27-H (∼15 µM), consistent with an impaired Ca2+-desensitization effect of the mutant peptide on skinned muscle strips. Upon binding to αTm, HcTnI-C27 acquired a weakly stable helix-like conformation involving residues near Trp191, as shown by transferred nuclear Overhauser effect spectroscopy and hydrogen/deuterium exchange experiments. With the potent Ca2+-desensitization effect of HcTnI-C27 on skinned cardiac muscle from a mouse model of hypertrophic cardiomyopathy, the data support that the C-terminal end domain of TnI can function as an isolated peptide with the intrinsic capacity of binding tropomyosin, providing a promising therapeutic approach to selectively improve diastolic function of the heart.


Assuntos
Cardiomiopatia Hipertrófica/genética , Fibras Musculares Esqueléticas/metabolismo , Miofibrilas/metabolismo , Peptídeos/química , Tropomiosina/metabolismo , Troponina I/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cálcio/metabolismo , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/patologia , Cardiomiopatia Hipertrófica/prevenção & controle , Modelos Animais de Doenças , Expressão Gênica , Humanos , Cinética , Camundongos , Simulação de Acoplamento Molecular , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Relaxamento Muscular , Mutação , Miofibrilas/efeitos dos fármacos , Miofibrilas/patologia , Peptídeos/genética , Peptídeos/metabolismo , Peptídeos/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Tropomiosina/química , Tropomiosina/genética , Troponina I/genética , Troponina I/metabolismo
17.
J Mol Biol ; 433(9): 166901, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33647290

RESUMO

Striated muscle responds to mechanical overload by rapidly up-regulating the expression of the cardiac ankyrin repeat protein, CARP, which then targets the sarcomere by binding to titin N2A in the I-band region. To date, the role of this interaction in the stress response of muscle remains poorly understood. Here, we characterise the molecular structure of the CARP-receptor site in titin (UN2A) and its binding of CARP. We find that titin UN2A contains a central three-helix bundle fold (ca 45 residues in length) that is joined to N- and C-terminal flanking immunoglobulin domains by long, flexible linkers with partial helical content. CARP binds titin by engaging an α-hairpin in the three-helix fold of UN2A, the C-terminal linker sequence, and the BC loop in Ig81, which jointly form a broad binding interface. Mutagenesis showed that the CARP/N2A association withstands sequence variations in titin N2A and we use this information to evaluate 85 human single nucleotide variants. In addition, actin co-sedimentation, co-transfection in C2C12 cells, proteomics on heart lysates, and the mechanical response of CARP-soaked myofibrils imply that CARP induces the cross-linking of titin and actin myofilaments, thereby increasing myofibril stiffness. We conclude that CARP acts as a regulator of force output in the sarcomere that preserves muscle mechanical performance upon overload stress.


Assuntos
Actinas/química , Actinas/metabolismo , Conectina/química , Conectina/metabolismo , Proteínas Musculares/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Reagentes para Ligações Cruzadas/química , Reagentes para Ligações Cruzadas/metabolismo , Masculino , Camundongos , Proteínas Musculares/química , Proteínas Musculares/genética , Mutação , Miofibrilas/química , Miofibrilas/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Maleabilidade , Ligação Proteica , Coelhos , Proteínas Repressoras/química , Proteínas Repressoras/genética , Sarcômeros/química , Sarcômeros/metabolismo
18.
BMC Cardiovasc Disord ; 21(1): 125, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33663396

RESUMO

BACKGROUND: Right ventricular dysfunction after CABG is associated with poor peri- and postoperative outcomes. We aimed to identify clinical and experimental predictors for preoperative inapparent right ventricular dysfunction and therefore hypothesized that reduced myofilament force development as well as altered levels of biomarkers might predict inapparent right ventricular dysfunction. METHODS: From 08/2016 to 02/2018, 218 patients scheduled for CABG were divided into two groups (TAPSE ≥ 20 mm, n = 178; TAPSE < 20 mm, n = 40). Baseline serum samples for biomarkers (Galectin, TGFß1, N Acyl-SDMA, Arginine, ADMA and Pentraxin-3), clinical laboratory and transthoracic echocardiographic parameters were evaluated. To examine the myocardial apparatus of the right ventricle intraoperative right auricular tissue was harvested for stepwise skinned fiber force measurements. RESULTS: Patients with TAPSE < 20 mm had a higher incidence of DM (55 vs. 34%, p = 0.018), preoperative AFib (43 vs. 16%, p < 0.001), reduced GFR (67 ± 18 vs. 77 ± 24 ml/min/1.73 m2, p = 0.013), larger LA area (22 ± 6 vs. 20 ± 5 cm2, p = 0.005) and reduced LVEF (50 vs. 55%, p = 0.008). Furthermore, higher serum ADMA (0.70 ± 0.13 vs. 0.65 ± 0.15 µmol/l, p = 0.046) and higher serum Pentraxin-3 levels (3371 ± 1068 vs. 2681 ± 1353 pg/dl, p = 0.004) were observed in these patients. Skinned fiber force measurements showed significant lower values at almost every step of calcium concentration (pCa 4.52 to pCa 5.5, p < 0.01 and pCa 5.75-6.0, p < 0.05). Multivariable analysis revealed DM (OR 2.53, CI 1.12-5.73, Euro Score II (OR 1.34, CI 1.02-1.78), preoperative AF (OR 4.86, CI 2.06-11.47), GFR (OR 7.72, CI 1.87-31.96), albumin (OR 1.56, CI 0.52-2.60), Pentraxin-3 (OR 19.68, CI 14.13-25.24), depressed LVEF (OR 8.61, CI 6.37-10.86), lower force values: (pCa 5.4; OR 2.34, CI 0.40-4.29 and pCa 5.2; OR 2.00, CI 0.39-3.60) as predictors for clinical inapparent right heart dysfunction. CONCLUSIONS: These preliminary data showed that inapparent right heart dysfunction in CAD is already associated with reduced force development of the contractile apparatus.


Assuntos
Cálcio/metabolismo , Ponte de Artéria Coronária/efeitos adversos , Doença da Artéria Coronariana/cirurgia , Contração Miocárdica , Miofibrilas/metabolismo , Disfunção Ventricular Direita/etiologia , Função Ventricular Direita , Idoso , Arginina/análogos & derivados , Arginina/sangue , Doenças Assintomáticas , Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/fisiopatologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Albumina Sérica Humana/metabolismo , Componente Amiloide P Sérico/metabolismo , Resultado do Tratamento , Disfunção Ventricular Direita/sangue , Disfunção Ventricular Direita/diagnóstico por imagem , Disfunção Ventricular Direita/fisiopatologia
19.
Commun Biol ; 4(1): 365, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33742108

RESUMO

The precise assembly of actin-based thin filaments is crucial for muscle contraction. Dysregulation of actin dynamics at thin filament pointed ends results in skeletal and cardiac myopathies. Here, we discovered adenylyl cyclase-associated protein 2 (CAP2) as a unique component of thin filament pointed ends in cardiac muscle. CAP2 has critical functions in cardiomyocytes as it depolymerizes and inhibits actin incorporation into thin filaments. Strikingly distinct from other pointed-end proteins, CAP2's function is not enhanced but inhibited by tropomyosin and it does not directly control thin filament lengths. Furthermore, CAP2 plays an essential role in cardiomyocyte maturation by modulating pre-sarcomeric actin assembly and regulating α-actin composition in mature thin filaments. Identification of CAP2's multifunctional roles provides missing links in our understanding of how thin filament architecture is regulated in striated muscle and it reveals there are additional factors, beyond Tmod1 and Lmod2, that modulate actin dynamics at thin filament pointed ends.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Transporte/metabolismo , Desenvolvimento Muscular , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Fatores Etários , Animais , Proteínas de Transporte/genética , Células Cultivadas , Embrião de Galinha , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos Sprague-Dawley , Transdução de Sinais , Tropomiosina/metabolismo
20.
Int J Sport Nutr Exerc Metab ; 31(3): 209-216, 2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33601335

RESUMO

The impact of resistance exercise frequency on muscle protein synthesis rates remains unknown. The aim of this study was to compare daily myofibrillar protein synthesis rates over a 7-day period of low-frequency (LF) versus high-frequency (HF) resistance exercise training. Nine young men (21 ± 2 years) completed a 7-day period of habitual physical activity (BASAL). This was followed by a 7-day exercise period of volume-matched, LF (10 × 10 repetitions at 70% one-repetition maximum, once per week) or HF (2 × 10 repetitions at ∼70% one-repetition maximum, five times per week) resistance exercise training. The participants had one leg randomly allocated to LF and the other to HF. Skeletal muscle biopsies and daily saliva samples were collected to determine myofibrillar protein synthesis rates using 2H2O, with intracellular signaling determined using Western blotting. The myofibrillar protein synthesis rates did not differ between the LF (1.46 ± 0.26%/day) and HF (1.48 ± 0.33%/day) conditions over the 7-day exercise training period (p > .05). There were no significant differences between the LF and HF conditions over the first 2 days (1.45 ± 0.41%/day vs. 1.25 ± 0.46%/day) or last 5 days (1.47 ± 0.30%/day vs. 1.50 ± 0.41%/day) of the exercise training period (p > .05). Daily myofibrillar protein synthesis rates were not different from BASAL at any time point during LF or HF (p > .05). The phosphorylation status and total protein content of selected proteins implicated in skeletal muscle ribosomal biogenesis were not different between conditions (p > .05). Under the conditions of the present study, resistance exercise training frequency did not modulate daily myofibrillar protein synthesis rates in young men.


Assuntos
Proteínas Musculares/biossíntese , Miofibrilas/metabolismo , Treinamento de Força , Actigrafia/estatística & dados numéricos , Biópsia , Óxido de Deutério/metabolismo , Dieta , Ingestão de Energia , Humanos , Perna (Membro) , Masculino , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Fosforilação , Distribuição Aleatória , Proteínas Ribossômicas/biossíntese , Transdução de Sinais , Fatores de Tempo , Adulto Jovem
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