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1.
J Photochem Photobiol B ; 202: 111680, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31810038

RESUMO

Tissue engineering and stem cell rehabilitation are the hopeful aspects that are being investigated for the management of Myocardial Infarction (MI); cardiac patches have been used to start myocardial rejuvenation. In this study, we engineered p-phenylenediamine surface functionalized (modif-CQD) into the Silk fibroin/PLA (SF/PLA) nanofibrous bioactive scaffolds with improved physico-chemical abilities, mechanical and cytocompatibility to cardiomyocytes. The micrograph results visualized the morphological improved spherical modif-CQD have been equivalently spread throughout the SF/PLA bioactive cardiac scaffolds. The fabricated CQD@SF/PLA nanofibrous bioactive scaffolds were highly porous with fully consistent pores; effectively improved young modulus and swelling asset for the suitability and effective implantation efficacy. The scaffolds were prepared with rat cardiomyocytes and cultured for up to 7 days, without electrical incentive. After 7 days of culture, the scaffold pores all over the construct volume were overflowing with cardiomyocytes. The metabolic activity and viability of the cardiomyocytes in CQD@SF/PLA scaffolds were significantly higher than cardiomyocytes in Silk fibroin /PLA scaffolds. The integration of CQD also influenced greatly and increases the expression of cardiac-marker genes. The results of the present investigations evidently recommended that well-organized cardiac nanofibrous scaffold with greater cardiac related mechanical abilities and biocompatibilities for cardiac tissue engineering and nursing care applications.


Assuntos
Fibroínas/química , Nanofibras/química , Pontos Quânticos/química , Engenharia Tecidual , Tecidos Suporte/química , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Carbono/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Raios Infravermelhos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Nanofibras/toxicidade , Poliésteres/química , RNA Mensageiro/metabolismo , Ratos , Troponina C/genética , Troponina C/metabolismo
2.
Food Chem ; 307: 125645, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31644983

RESUMO

A simple electrochemical sensor was developed to determine the concentration of Ca2+ in meat. Graphene was treated with oxygen plasma for 10 s and 30 s comparing with the pristine graphene. Through analyzing morphology and chemical composition, the graphene with the lowest defect density was chosen to mix with bovine serum albumin molecule-functionalized gold nanoparticles. It was interesting that only a few gold nanoparticles were trapped in the graphene with 10 s plasma treatment. Then, under the optimal condition measured, the limit of detection was detected as 3.9 × 10-8 M with a linear relationship from 5 × 10-8 to 3 × 10-4 M. Finally, the proposed electrochemical method was applied to detect Ca2+ in the pork sample with stability and reproducibility verified by parallel detections. Thus, the proposed method demonstrates its potential for effectively detecting Ca2+ in meat and prominently reduces time consumption on operations and pretreatments.


Assuntos
Técnicas Biossensoriais/instrumentação , Cálcio na Dieta/análise , Técnicas Eletroquímicas/métodos , Carne/análise , Nanopartículas Metálicas/química , Técnicas Biossensoriais/métodos , Cálcio na Dieta/metabolismo , Ouro/química , Grafite/química , Limite de Detecção , Reprodutibilidade dos Testes , Troponina C/metabolismo
3.
Biotechnol J ; 14(8): e1800725, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30927511

RESUMO

Cardiomyocytes (CMs) generated from human pluripotent stem cells (hPSCs) are immature in their structure and function, limiting their potential in disease modeling, drug screening, and cardiac cellular therapies. Prior studies have demonstrated that coculture of hPSC-derived CMs with other cardiac cell types, including endothelial cells (ECs), can accelerate CM maturation. To address whether the CM differentiation stage at which ECs are introduced affects CM maturation, the authors coculture hPSC-derived ECs with hPSC-derived cardiac progenitor cells (CPCs) and CMs and analyze the molecular and functional attributes of maturation. ECs have a more significant effect on acceleration of maturation when cocultured with CPCs than with CMs. EC coculture with CPCs increases CM size, expression of sarcomere, and ion channel genes and proteins, the presence of intracellular membranous extensions, and chronotropic response compared to monoculture. Maturation is accelerated with an increasing EC:CPC ratio. This study demonstrates that EC incorporation at the CPC stage of CM differentiation expedites CM maturation, leading to cells that may be better suited for in vitro and in vivo applications of hPSC-derived CMs.


Assuntos
Técnicas de Cocultura/métodos , Células Endoteliais/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Células-Tronco Pluripotentes/citologia , Agonistas Adrenérgicos beta/farmacologia , Diferenciação Celular , Tamanho Celular , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Isoproterenol/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Troponina C/metabolismo , Troponina I/metabolismo
4.
PLoS Comput Biol ; 15(3): e1006712, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30845143

RESUMO

Skeletal muscle contraction is initiated when an action potential triggers the release of Ca2+ into the sarcomere in a process referred to as excitation-contraction coupling. The speed and scale of this process makes direct observation very challenging and invasive. To determine how the concentration of Ca2+ changes within the myofibril during a single activation, several simulation models have been developed. These models follow a common pattern; divide the half sarcomere into a series of compartments, then use ordinary differential equations to solve reactions occurring within and between the compartments. To further develop this type of simulation, we have created a realistic structural model of a skeletal muscle myofibrillar half-sarcomere using MCell software that incorporates the myofilament lattice structure. Using this simulation model, we were successful in reproducing the averaged calcium transient during a single activation consistent with both the experimental and previous simulation results. In addition, our simulation demonstrated that the inclusion of the myofilament lattice within our model produced an asymmetric distribution of Ca2+, with more Ca2+ accumulating near the Z-disk and less Ca2+ reaching the m-line. This asymmetric distribution of Ca2+ is also apparent when we examine how the Ca2+ are bound to the troponin-C proteins along the actin filaments. Our simulation model also allowed us to produce advanced visualizations of this process, including two simulation animations, allowing us to view Ca2+ release, diffusion, binding and uptake within the myofibrillar half-sarcomere.


Assuntos
Cálcio/metabolismo , Modelos Biológicos , Músculo Esquelético/metabolismo , Sarcômeros , Processos Estocásticos , Trifosfato de Adenosina/metabolismo , Animais , Método de Monte Carlo , Troponina C/metabolismo
5.
Biochim Biophys Acta Mol Cell Res ; 1866(7): 1046-1053, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30716407

RESUMO

This review aims at giving a rational frame to understand the diversity of EF hand containing calcium binding proteins and their roles, with special focus on three members of this huge protein family, namely calmodulin, troponin C and parvalbumin. We propose that these proteins are members of structured macromolecular complexes, termed calcisomes, which constitute building devices allowing treatment of information within eukaryotic cells and namely calcium signals encoding and decoding, as well as control of cytosolic calcium levels in resting cells. Calmodulin is ubiquitous, present in all eukaryotic cells, and pleiotropic. This may be explained by its prominent role in regulating calcium movement in and out of the cell, thus maintaining calcium homeostasis which is fundamental for cell survival. The protein is further involved in decoding transient calcium signals associated with calcium movements after cell stimulation. We will show that the specificity of calmodulin's actions may be more easily explained if one considers its role in the light of calcisomes. Parvalbumin should not be considered as a simple intracellular calcium buffer. It is also a key factor for regulating calcium homeostasis in specific cells that need a rapid retrocontrol of calcium transients, such as fast muscle fibers. Finally, we propose that troponin C, with its four calcium binding domains distributed between two lobes presenting different calcium binding kinetics, exhibits all the characteristics needed to trigger and then post modulate muscle contraction and thus appears as a typical Feed Forward Loop system. If the present conjectures prove accurate, the way will be paved for a new pharmacology targeting the cell calcium signaling machinery. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Calmodulina/metabolismo , Parvalbuminas/metabolismo , Troponina C/metabolismo , Animais , Humanos
6.
Biochemistry ; 58(7): 908-917, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30620548

RESUMO

The key events in regulating muscle contraction involve the troponin (Tn) heterotrimeric protein complex in which the binding to and release of Ca2+ from the highly conserved troponin C (TnC) subunit trigger a series of structural changes within Tn, and the other thin filament proteins, to result in contraction. In the heart, the control of contraction and relaxation events can be altered by many single-point mutations that may result in cardiomyopathy and sometimes sudden cardiac death. Here we have examined the structural effects of one hypertrophic cardiomyopathy mutation, L29Q, on Ca2+-induced structural transitions within whole TnC. This mutation is of particular interest as several physiological and structural studies have indicated that the response of TnC to Ca2+ binding is altered in the presence of the L29Q mutation, but the structural nature of these changes continues to be debated. In addition, little is known about the effect of this mutation in the Ca2+ free state. Here we have used paramagnetic relaxation enhancement nuclear magnetic resonance (PRE-NMR) to assess the structural effects arising from the L29Q mutation. PRE-NMR distances obtained from a nitroxide spin-label at Cys84 showed that the L29Q mutation perturbs the structure of the TnC N-domain in the presence and absence of Ca2+, with a more "open" TnC N-domain observed in the apo form. In addition, binding of Ca2+ to the TnC-L29Q construct triggers a change in the orientation between the two domains of TnC. Together, these structural perturbations, revealed by PRE-NMR, provide insight into the pathogenesis of this mutation.


Assuntos
Cardiomiopatia Hipertrófica/genética , Leucina/genética , Mutação , Troponina C/química , Troponina C/genética , Animais , Cálcio/metabolismo , Cisteína/química , Cisteína/genética , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Ratos , Marcadores de Spin , Troponina C/metabolismo
7.
Arch Biochem Biophys ; 663: 95-100, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30584890

RESUMO

The cardiac contraction-relaxation cycle is controlled by a sophisticated set of machinery. Of particular interest, is the revelation that allosteric networks transmit effects of binding at one site to influence troponin complex dynamics and structural-mediated signaling in often distal, functional sites in the myofilament. Our recent observations provide compelling evidence that allostery can explain the function of large-scale macromolecular events. Here we elaborate on our recent findings of interdomain communication within troponin C, using cutting-edge structural biology approaches, and highlight the importance of unveiling the unknown, distant communication networks within this system to obtain more comprehensive knowledge of how allostery impacts cardiac physiology and disease.


Assuntos
Troponina C/metabolismo , Troponina I/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Animais , Humanos , Ligação Proteica , Relação Estrutura-Atividade , Troponina C/química , Troponina I/química
8.
Sci Rep ; 8(1): 16084, 2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30382112

RESUMO

Dynamic movements of the cardiac troponin complex are an important component of the cardiac cycle. Whether cardiac troponins are subjected to irreversible advanced glycation end-product (AGE) modification is unknown. This study interrogated human and rat cardiac troponin-C, troponin-I and troponin-T to identify endogenous AGE modifications using mass spectrometry (LC-MS/MS). AGE modifications were detected on two amino acid residues of human troponin-C (Lys6, Lys39), thirteen troponin-I residues (Lys36, Lys50, Lys58, Arg79, Lys117, Lys120, Lys131, Arg148, Arg162, Lys164, Lys183, Lys193, Arg204), and three troponin-T residues (Lys107, Lys125, Lys227). AGE modifications of three corresponding troponin-I residues (Lys58, Lys120, Lys194) and two corresponding troponin-T residues (Lys107, Lys227) were confirmed in cardiac tissue extracts from an experimental rodent diabetic model. Additionally, novel human troponin-I phosphorylation sites were detected (Thr119, Thr123). Accelerated AGE modification of troponin-C was evident in vitro with hexose sugar exposure. This study provides the first demonstration of the occurrence of cardiac troponin complex AGE-modifications. These irreversible AGE modifications are situated in regions of the troponin complex known to be important in myofilament relaxation, and may be of particular pathological importance in the pro-glycation environment of diabetic cardiomyopathy.


Assuntos
Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/complicações , Cardiomiopatias Diabéticas/patologia , Produtos Finais de Glicação Avançada/metabolismo , Coração/fisiologia , Troponina C/metabolismo , Animais , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/metabolismo , Glicosilação , Humanos , Masculino , Ratos , Ratos Sprague-Dawley
9.
Biochemistry ; 57(46): 6461-6469, 2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30376637

RESUMO

Compounds that directly modulate the affinity of the thin filament calcium regulatory proteins in cardiac muscle have potential for treating heart disease. A recent "proof of concept" study showed that the desensitizer W7 can correct hyper-calcium-sensitive sarcomeres from RCM R193H inhibitory subunit troponin I (cTnI) transgenic mice. We have determined the high-resolution nuclear magnetic resonance solution structure of W7 bound to the regulatory domain of calcium binding subunit troponin C (cNTnC)-cTnI cChimera designed to represent the key aspects of the cTnC-cTnI interface. The structure shows that W7 does not perturb the overall structure of the cTnC-cTnI interface, with the helical structure and position of the cTnI switch region remaining intact upon W7 binding. The naphthalene ring of W7 sits in the hydrophobic pocket created by the cNTnC-cTnI switch peptide interface, while the positively charged amine tail extends into the solvent. The positively charged tail of W7 is in the proximity of Arg147 of the cTnI switch region, supporting the suggestion that electrostatic repulsion is an aspect underlying the mechanism of desensitization. Ser84 (replacing the unique Cys84 in cTnC reported to make a reversible covalent bond with levosimendan) also contacts W7.


Assuntos
Cálcio/metabolismo , Inibidores Enzimáticos/metabolismo , Sulfonamidas/metabolismo , Troponina C/metabolismo , Animais , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica
10.
Cell Biol Int ; 42(11): 1556-1563, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30095216

RESUMO

Cardiovascular disease is a leading cause of death worldwide, requiring the development of new therapeutic strategies including stem cell therapy. Pentraxins (PTXs) are a superfamily of proteins highly involved in different myocardial disorders, and thus this study aimed to identify the modulation of long pentraxin 3 (PTX3) in the differentiation of mouse embryonic stem cells (mESCs) toward cardiomyocytes. Cell toxicity of PTX3 was detected by MTT and LDH assays in mESCs. Embryoid bodies (EBs) were differentiated using hanging drop method, and the beating was observed under microscope. Expressional levels of early cardiac progenitor marker genes were assessed by qRT-PCR. Expression of marker proteins in early myocardial development and the activation of JNK signaling pathway was evaluated by Western blot. PTX3 treatment at 50 ng/mL significantly promoted the expression of cardiac-specific marker genes including Nkx2.5, Mef2c, Tbx5, dHand, and αMHC, and increased the expression of cardiac maturity indicative markers including connexin 43 and troponin C1. PTX3 enhanced the phosphorylation of JNK across the incubation duration, whereas the activation of p38 remained the same as control group. Co-treatment of JNK signaling pathway inhibitor SP600125 impaired the PTX3-promoted transcription of Nkx2.5, Mef2c, Tbx5, dHand, and αMHC. This study revealed the promotion of PTX3 in the differentiation of mESCs into cardiomyocytes and the underlying mechanism.


Assuntos
Proteína C-Reativa/farmacologia , Diferenciação Celular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Miocárdio/citologia , Componente Amiloide P Sérico/farmacologia , Animais , Biomarcadores/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Conexina 43/metabolismo , Corpos Embrioides/citologia , Corpos Embrioides/efeitos dos fármacos , Corpos Embrioides/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Especificidade de Órgãos , Troponina C/metabolismo
11.
Am J Physiol Heart Circ Physiol ; 315(5): H1453-H1462, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30141984

RESUMO

Action potential duration (APD) alternans (APD-ALT), defined as beat-to-beat oscillations in APD, has been proposed as an important clinical marker for chronic atrial fibrillation (cAF) risk when it occurs at pacing rates of 120-200 beats/min. Although the ionic mechanisms for occurrence of APD-ALT in human cAF at these clinically relevant rates have been investigated, little is known about the effects of myofilament protein kinetics on APD-ALT. Therefore, we used computer simulations of single cell function to explore whether remodeling in myofilament protein kinetics in human cAF alters the occurrence of APD-ALT and to uncover how these mechanisms are affected by sarcomere length and the degree of cAF-induced myofilament remodeling. Mechanistically based, bidirectionally coupled electromechanical models of human right and left atrial myocytes were constructed, incorporating both ionic and myofilament remodeling associated with cAF. By comparing results from our electromechanical model with those from the uncoupled ionic model, we found that intracellular Ca2+ concentration buffering of troponin C has a dampening effect on the magnitude of APD-ALT (APD-ANM) at slower rates (150 beats/min) due to the cooperativity between strongly bound cross-bridges and Ca2+-troponin C binding affinity. We also discovered that cAF-induced enhanced thin filament activation enhanced APD-ANM at these clinically relevant heart rates (150 beats/min). In addition, longer sarcomere lengths increased APD-ANM, suggesting that atrial stretch is an important modulator of APD-ALT. Together, these findings demonstrate that myofilament kinetics mechanisms play an important role in altering APD-ALT in human cAF. NEW & NOTEWORTHY Using a single cell simulation approach, we explored how myofilament protein kinetics alter the formation of alternans in action potential duration (APD) in human myocytes with chronic atrial fibrillation remodeling. We discovered that enhanced thin filament activation and longer sarcomere lengths increased the magnitude of APD alternans at clinically important pacing rates of 120-200 beats/min. Furthermore, we found that altered intracellular Ca2+ concentration buffering of troponin C has a dampening effect on the magnitude of APD alternans.


Assuntos
Potenciais de Ação , Fibrilação Atrial/metabolismo , Sinalização do Cálcio , Átrios do Coração/metabolismo , Frequência Cardíaca , Modelos Cardiovasculares , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Fibrilação Atrial/fisiopatologia , Doença Crônica , Simulação por Computador , Acoplamento Excitação-Contração , Átrios do Coração/fisiopatologia , Humanos , Cinética , Contração Miocárdica , Sarcômeros/metabolismo , Troponina C/metabolismo
12.
J Mol Cell Cardiol ; 123: 26-37, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30138628

RESUMO

Acto-myosin cross-bridge kinetics are important for beat-to-beat regulation of cardiac contractility; however, physiological and pathophysiological mechanisms for regulation of contractile kinetics are incompletely understood. Here we explored whether thin filament-mediated Ca2+ sensitization influences cross-bridge kinetics in permeabilized, osmotically compressed cardiac muscle preparations. We used a murine model of hypertrophic cardiomyopathy (HCM) harboring a cardiac troponin C (cTnC) Ca2+-sensitizing mutation, Ala8Val in the regulatory N-domain. We also treated wild-type murine muscle with bepridil, a cTnC-targeting Ca2+ sensitizer. Our findings suggest that both methods of increasing myofilament Ca2+ sensitivity increase cross-bridge cycling rate measured by the rate of tension redevelopment (kTR); force per cross-bridge was also enhanced as measured by sinusoidal stiffness and I1,1/I1,0 ratio from X-ray diffraction. Computational modeling suggests that Ca2+ sensitization through this cTnC mutation or bepridil accelerates kTR primarily by promoting faster cross-bridge detachment. To elucidate if myofilament structural rearrangements are associated with changes in kTR, we used small angle X-ray diffraction to simultaneously measure myofilament lattice spacing and isometric force during steady-state Ca2+ activations. Within in vivo lattice dimensions, lattice spacing and steady-state isometric force increased significantly at submaximal activation. We conclude that the cTnC N-domain controls force by modulating both the number and rate of cycling cross-bridges, and that the both methods of Ca2+ sensitization may act through stabilization of cTnC's D-helix. Furthermore, we propose that the transient expansion of the myofilament lattice during Ca2+ activation may be an additional factor that could increase the rate of cross-bridge cycling in cardiac muscle. These findings may have implications for the pathophysiology of HCM.


Assuntos
Cálcio/metabolismo , Contração Miocárdica , Miocárdio/metabolismo , Miofibrilas/metabolismo , Troponina C/metabolismo , Algoritmos , Animais , Sinalização do Cálcio , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/patologia , Cardiomiopatia Hipertrófica/fisiopatologia , Modelos Animais de Doenças , Feminino , Humanos , Contração Isométrica , Cinética , Masculino , Camundongos , Modelos Moleculares , Modelos Teóricos , Mutação , Miocárdio/química , Miofibrilas/química , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Troponina C/química , Troponina C/genética
13.
J Physiol ; 596(19): 4651-4663, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29992562

RESUMO

KEY POINTS: The Ca2+ -desensitizing D73N mutation in slow skeletal/cardiac troponin C caused dilatated cardiomyopathy in mice, but the consequences of this mutation in skeletal muscle were not known. The D73N mutation led to a rightward shift in the force versus pCa (-log [Ca]) relationship in slow-twitch mouse fibres. The D73N mutation led to a rightward shift in the force-stimulation frequency relationship and reduced fatigue resistance of mouse soleus muscle. The D73N mutation led to reduced cross-sectional area of slow-twitch fibres in mouse soleus muscle without affecting fibre type composition of the muscle. The D73N mutation resulted in significantly shorter times to peak force and to relaxation during isometric twitches and tetani in mouse soleus muscle. The D73N mutation led to major changes in physiological properties of mouse soleus muscle, converting slow muscle toward a fast muscle phenotype. ABSTRACT: The missense mutation, D73N, in mouse cardiac troponin C has a profound impact on cardiac function, mediated by a decreased myofilament Ca2+ sensitivity. Mammalian cardiac muscle and slow skeletal muscle normally share expression of the same troponin C isoform. Therefore, the objective of this study was to determine the consequences of the D73N mutation in skeletal muscle, as a potential mechanism that contributes to the morbidity associated with heart failure or other conditions in which Ca2+ sensitivity might be altered. Effects of the D73N mutation on physiological properties of mouse soleus muscle, in which slow-twitch fibres are prevalent, were examined. The mutation resulted in a rightward shift of the force-stimulation frequency relationship, and significantly faster kinetics of isometric twitches and tetani in isolated soleus muscle. Furthermore, soleus muscles from D73N mice underwent a significantly greater reduction in force during a fatigue test. The mutation significantly reduced slow fibre mean cross-sectional area without affecting soleus fibre type composition. The effects of the mutation on Ca2+ sensitivity of force development in soleus skinned slow and fast fibres were also examined. As expected, the D73N mutation did not affect the Ca2+ sensitivity of force development in fast fibres but resulted in substantially decreased Ca2+ sensitivity in slow fibres. The results demonstrate that a point mutation in a single constituent of myofilaments (slow/cardiac troponin C) led to major changes in physiological properties of skeletal muscle and converted slow muscle toward a fast muscle phenotype with reduced fatigue resistance and Ca2+ sensitivity of force generation.


Assuntos
Hormônios e Agentes Reguladores de Cálcio/farmacologia , Cálcio/farmacologia , Contração Muscular , Fibras Musculares de Contração Rápida/fisiologia , Fibras Musculares de Contração Lenta/fisiologia , Músculo Esquelético/fisiologia , Troponina C/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares de Contração Rápida/efeitos dos fármacos , Fibras Musculares de Contração Lenta/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Mutação , Fenótipo , Troponina C/genética
14.
J Comp Physiol B ; 188(5): 863-876, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30039299

RESUMO

To determine whether the regulation of calpain system is involved in non-hibernators and hibernators in disused condition, the soleus (SOL) and extensor digitorum longus (EDL) muscles were used for investigating the muscle mass, the ratio of muscle wet weight/body weight (MWW/BW), fiber-type distribution, fiber cross-sectional area (CSA), and the protein expression of MuRF1, calpain-1, calpain-2, calpastatin, desmin, troponin T, and troponin C in hindlimb unloading rats and hibernating Daurian ground squirrels. The muscle mass, MWW/BW, and fiber CSA were found significantly decreased in SOL and EDL of hindlimb unloading rats, but unchanged in hibernating ground squirrels. The MuRF1 expression was increased in both SOL and EDL of unloading rats, while it was only increased in SOL, but maintained in EDL of hibernating ground squirrels. The expression levels of calpain-1 and calpain-2 were increased in different degrees in unloaded SOL and EDL in rats, while they were maintained in EDL and even reduced in SOL of hibernating ground squirrels. Besides, the expression of calpastatin was decreased in unloaded rats, but increased in hibernating ground squirrels. The desmin expression was decreased in unloaded rats, but maintained in hibernating squirrels. Interestingly, the levels of troponin T and troponin C were decreased in both SOL and EDL of unloaded rats, but increased in hibernating ground squirrels with muscle-type specificity. In conclusion, differential calpain activation and substrate-selective degradation in slow and fast muscles are involved in the mechanisms of muscle atrophy of unloaded rats and remarkable ability of muscle maintenance of hibernating ground squirrels.


Assuntos
Calpaína/metabolismo , Músculo Esquelético/metabolismo , Sciuridae/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Desmina/metabolismo , Feminino , Hibernação , Membro Posterior/fisiologia , Proteínas Musculares/metabolismo , Ratos Sprague-Dawley , Proteínas com Motivo Tripartido/metabolismo , Troponina C/metabolismo , Troponina T/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
15.
Biophys J ; 114(7): 1646-1656, 2018 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-29642034

RESUMO

The heterotrimeric cardiac troponin complex is a key regulator of contraction and plays an essential role in conferring Ca2+ sensitivity to the sarcomere. During ischemic injury, rapidly accumulating protons acidify the myoplasm, resulting in markedly reduced Ca2+ sensitivity of the sarcomere. Unlike the adult heart, sarcomeric Ca2+ sensitivity in fetal cardiac tissue is comparatively pH insensitive. Replacement of the adult cardiac troponin I (cTnI) isoform with the fetal troponin I (ssTnI) isoform renders adult cardiac contractile machinery relatively insensitive to acidification. Alignment and functional studies have determined histidine 132 of ssTnI to be the predominant source of this pH insensitivity. Substitution of histidine at the cognate position 164 in cTnI confers the same pH insensitivity to adult cardiac myocytes. An alanine at position 164 of cTnI is conserved in all mammals, with the exception of the platypus, which expresses a proline. Prolines are biophysically unique because of their innate conformational rigidity and helix-disrupting function. To provide deeper structure-function insight into the role of the TnC-TnI interface in determining contractility, we employed a live-cell approach alongside molecular dynamics simulations to ascertain the chemo-mechanical implications of the disrupted helix 4 of cTnI where position 164 exists. This important motif belongs to the critical switch region of cTnI. Substitution of a proline at position 164 of cTnI in adult rat cardiac myocytes causes increased contractility independent of alterations in the Ca2+ transient. Free-energy perturbation calculations of cTnC-Ca2+ binding indicate no difference in cTnC-Ca2+ affinity. Rather, we propose the enhanced contractility is derived from new salt bridge interactions between cTnI helix 4 and cTnC helix A, which are critical in determining pH sensitivity and contractility. Molecular dynamics simulations demonstrate that cTnI A164P structurally phenocopies ssTnI under baseline but not acidotic conditions. These findings highlight the evolutionarily directed role of the TnI-cTnC interface in determining cardiac contractility.


Assuntos
Contração Miocárdica , Troponina C/química , Troponina C/metabolismo , Troponina I/química , Troponina I/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Ratos , Ratos Sprague-Dawley
16.
Biochemistry ; 57(15): 2256-2265, 2018 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-29558109

RESUMO

The development of calcium sensitizers for the treatment of systolic heart failure presents difficulties, including judging the optimal efficacy and the specificity to target cardiac muscle. The thin filament is an attractive target because cardiac troponin C (cTnC) is the site of calcium binding and the trigger for subsequent contraction. One widely studied calcium sensitizer is levosimendan. We have recently shown that when a covalent cTnC-levosimendan analogue is exchanged into cardiac muscle cells, they become constitutively active, demonstrating the potency of a covalent complex. We have also demonstrated that levosimendan reacts in vitro to form a reversible covalent thioimidate bond specifically with cysteine 84, unique to cTnC. In this study, we use mass spectrometry to show that the in vitro mechanism of action of levosimendan is consistent with an allosteric, reversible covalent inhibitor; to determine whether the presence of the cTnI switch peptide or changes in either Ca2+ concentration or pH modify the reaction kinetics; and to determine whether the reaction can occur with cTnC in situ in cardiac myofibrils. Using the derived kinetic rate constants, we predict the degree of covalently modified cTnC in vivo under the conditions studied. We observe that covalent bond formation would be highest under the acidotic conditions resulting from ischemia and discuss whether the predicted level could be sufficient to have therapeutic value. Irrespective of the in vivo mechanism of action for levosimendan, our results provide a rationale and basis for the development of reversible covalent drugs to target the failing heart.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Hidrazonas , Isquemia Miocárdica , Miofibrilas , Piridazinas , Troponina C , Animais , Cisteína/metabolismo , Humanos , Hidrazonas/química , Hidrazonas/farmacocinética , Hidrazonas/farmacologia , Concentração de Íons de Hidrogênio , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Miofibrilas/química , Miofibrilas/metabolismo , Miofibrilas/ultraestrutura , Piridazinas/química , Piridazinas/farmacocinética , Piridazinas/farmacologia , Simendana , Suínos , Troponina C/química , Troponina C/metabolismo
17.
BMC Biotechnol ; 18(1): 10, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29439686

RESUMO

BACKGROUND: The recently developed genetically encoded calcium indicator (GECI), called NTnC, has a novel design with reduced size due to utilization of the troponin C (TnC) as a Ca2+-binding moiety inserted into the mNeonGreen fluorescent protein. NTnC binds two times less Ca2+ ions while maintaining a higher fluorescence brightness at the basal level of Ca2+ in neurons as compared with the calmodulin-based GECIs, such as GCaMPs. In spite of NTnC's high brightness, pH-stability, and high sensitivity to single action potentials, it has a limited fluorescence contrast (F-Ca2+/F+Ca2+) and slow Ca2+ dissociation kinetics. RESULTS: Herein, we developed a new NTnC-like GECI with enhanced fluorescence contrast and kinetics by replacing the mNeonGreen fluorescent subunit of the NTnC indicator with EYFP. Similar to NTnC, the developed indicator, named iYTnC2, has an inverted fluorescence response to Ca2+ (i.e. becoming dimmer with an increase of Ca2+ concentration). In the presence of Mg2+ ions, iYTnC2 demonstrated a 2.8-fold improved fluorescence contrast in vitro as compared with NTnC. The iYTnC2 indicator has lower brightness and pH-stability, but similar photostability as compared with NTnC in vitro. Stopped-flow fluorimetry studies revealed that iYTnC2 has 5-fold faster Ca2+ dissociation kinetics than NTnC. When compared with GCaMP6f GECI, iYTnC2 has up to 5.6-fold faster Ca2+ association kinetics and 1.7-fold slower dissociation kinetics. During calcium transients in cultured mammalian cells, iYTnC2 demonstrated a 2.7-fold higher fluorescence contrast as compared with that for the NTnC. iYTnC2 demonstrated a 4-fold larger response to Ca2+ transients in neuronal cultures than responses of NTnC. iYTnC2 response in neurons was additionally characterized using whole-cell patch clamp. Finally, we demonstrated that iYTnC2 can visualize neuronal activity in vivo in the hippocampus of freely moving mice using a nVista miniscope. CONCLUSIONS: We demonstrate that expanding the family of NTnC-like calcium indicators is a promising strategy for the development of the next generation of GECIs with smaller molecule size and lower Ca2+ ions buffering capacity as compared with commonly used GECIs.


Assuntos
Cálcio/análise , Imagem Molecular/métodos , Neurônios/metabolismo , Proteínas Recombinantes/metabolismo , Troponina C/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Fluorescência , Fluorometria/métodos , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Masculino , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/instrumentação , Técnicas de Patch-Clamp , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Imagem com Lapso de Tempo , Troponina C/genética
18.
Mini Rev Med Chem ; 18(16): 1354-1362, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-27594341

RESUMO

Levosimendan is a pyridazinone-dinitrile derivative, emerged as a potent cardiotonic agent with dual inotropic and vasodilator activities in higher animals. This is a calcium (Ca2+) sensitizing cardiotonic agent, which has been shown to exert positive inotropic effects without increasing intracellular Ca2+ transient. This avoids Ca2+ overload that leads to arrhythmias and myocyte injuries, and do not increase the energy consumption for handling Ca2+ and has shown good activity against congestive heart failure (CHF), due to its increased myocardial contractility by stabilizing the calcium bound conformation of troponin C. Levosimendan also acts as a pulmonary and systemic vasodilator. The combination of positive inotropic and vasodilator activity has been beneficial in increasing cardiac output and decreasing left ventricular end-diastolic pressure, pulmonary wedge pressure, right atrial pressure, and systemic vascular resistance in CHF patients. The cardiac target protein of levosimendan and troponin C, is Ca2+-binding protein. This raises the possibility that levosimendan may interact with smooth muscle proteins, such as, calmodulin, and regulatory myosin light chains. Levosimendan relaxes coronary arteries and lowers Ca2+. The lowering of Ca2+ by levosimendan is consistent with opening of K+ channels and causes relaxation that is independent of Ca2+. However, most of the Ca2+ sensitizers may impair cardiac diastolic function as a result of increased Ca2+ sensitivity of the myofilaments. Levosimendan has not only improved the cardiac systolic function but also the diastolic relaxation in CHF.


Assuntos
Cálcio/metabolismo , Cardiotônicos/farmacologia , Hidrazonas/farmacologia , Piridazinas/farmacologia , Calmodulina/metabolismo , Cardiotônicos/metabolismo , Cardiotônicos/uso terapêutico , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Humanos , Hidrazonas/metabolismo , Hidrazonas/uso terapêutico , Relaxamento Muscular , Contração Miocárdica , Cadeias Leves de Miosina/metabolismo , Canais de Potássio/metabolismo , Piridazinas/metabolismo , Piridazinas/uso terapêutico , Simendana , Troponina C/metabolismo , Resistência Vascular
19.
Proc Natl Acad Sci U S A ; 114(51): 13453-13458, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29208709

RESUMO

Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt-bridge competition or "theft" mechanism that enables a phospho-triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf-1 to inhibiting G-protein-coupled receptor kinase 2 upon phosphorylation, thereby bridging MAP kinase and G-Protein-Coupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, competes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Structural elements underlying the theft occurred early in evolution and are found in 10% of homo-oligomers and 30% of hetero-oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding partners, the salt-bridge theft mechanism represents a facile strategy for promoting or disrupting protein interactions using solvent-accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change.


Assuntos
Sequência Conservada , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Substituição de Aminoácidos , Animais , Evolução Molecular , Humanos , Lisina/genética , Lisina/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/química , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Fosforilação , Ligação Proteica , Serina/genética , Serina/metabolismo , Troponina C/química , Troponina C/genética , Troponina C/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
20.
J Chem Inf Model ; 57(12): 3056-3069, 2017 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-29144742

RESUMO

Calcium-dependent cardiac muscle contraction is regulated by the protein complex troponin. Calcium binds to the N-terminal domain of troponin C (cNTnC) which initiates the process of contraction. Heart failure is a consequence of a disruption of this process. With the prevalence of this condition, a strong need exists to find novel compounds to increase the calcium sensitivity of cNTnC. Desirable are small chemical molecules that bind to the interface between cTnC and the cTnI switch peptide and exhibit calcium sensitizing properties by possibly stabilizing cTnC in an open conformation. To identify novel drug candidates, we employed a structure-based drug discovery protocol that incorporated the use of a relaxed complex scheme (RCS). In preparation for the virtual screening, cNTnC conformations were identified based on their ability to correctly predict known cNTnC binders using a receiver operating characteristics analysis. Following a virtual screen of the National Cancer Institute's Developmental Therapeutic Program database, a small number of molecules were experimentally tested using stopped-flow kinetics and steady-state fluorescence titrations. We identified two novel compounds, 3-(4-methoxyphenyl)-6,7-chromanediol (NSC600285) and 3-(4-methylphenyl)-7,8-chromanediol (NSC611817), that show increased calcium sensitivity of cTnC in the presence of the regulatory domain of cTnI. The effects of NSC600285 and NSC611817 on the calcium dissociation rate was stronger than that of the known calcium sensitizer bepridil. Thus, we identified a 3-phenylchromane group as a possible key pharmacophore in the sensitization of cardiac muscle contraction. Building on this finding is of interest to researchers working on development of drugs for calcium sensitization.


Assuntos
Cálcio/metabolismo , Cromanos/química , Cromanos/farmacologia , Desenho de Drogas , Troponina C/metabolismo , Projeto Auxiliado por Computador , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios Proteicos , Troponina C/química , Troponina I/química , Troponina I/metabolismo
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