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1.
Appl Biochem Biotechnol ; 189(2): 396-410, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31025171

RESUMO

Although embryonic stem (ES) cells (ESCs) may be a promising donor source for the repair of infarcted or ischemic heart tissues, their successful application in regenerative medicine has been hampered by difficulties in enriching, identifying, and selecting cardiomyocytes from the differentiating cells. We established transgenic human ES cell lines by transcriptional control of the α-cardiac myosin heavy chain (α-MHC) promoter driving green fluorescent protein (GFP) expression. Differentiated GFP-expressing cells display the characteristics of cardiomyocytes (CMs). Apela, a recently identified short peptide, up-regulated the expression of the cardiac-restricted transcription factors Tbx5 and GATA4 as well as differentiated the cardiomyocyte markers α-MHC and ß-MHC. Flow cytometric analysis showed that apela increased the percentage of GFP-expressing cells in the beating foci of the embryoid bodies. The percentage of cardiac troponin T (TNT)-positive cells and the protein expression of TNT were increased in the ES cell-derived CMs with apela treatment. Functionally, the contractile frequency of the ES-derived CMs responded appropriately to the vasoactive drugs isoprenaline and carbachol. Our work presented a protocol for specially labelling and enriching CMs by combining transgenic human ES cell lines and exogenous growth factor treatment.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias Humanas/metabolismo , Miócitos Cardíacos/metabolismo , Hormônios Peptídicos/metabolismo , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Linhagem Celular , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Humanos , Miócitos Cardíacos/citologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Hormônios Peptídicos/genética , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Troponina T/metabolismo , Regulação para Cima , Miosinas Ventriculares/genética , Miosinas Ventriculares/metabolismo
2.
Pflugers Arch ; 471(5): 701-717, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30767072

RESUMO

Several lines of evidence suggest that the primary effect of hypertrophic cardiomyopathy mutations in human ß-cardiac myosin is hypercontractility of the heart, which leads to subsequent hypertrophy, fibrosis, and myofilament disarray. Here, I describe three perspectives on the molecular basis of this hypercontractility. The first is that hypercontractility results from changes in the fundamental parameters of the actin-activated ß-cardiac myosin chemo-mechanical ATPase cycle. The second considers that hypercontractility results from an increase in the number of functionally accessible heads in the sarcomere for interaction with actin. The final and third perspective is that load dependence of contractility is affected by cardiomyopathy mutations and small-molecule effectors in a manner that changes the power output of cardiac contraction. Experimental approaches associated with each perspective are described along with concepts of therapeutic approaches that could prove valuable in treating hypertrophic cardiomyopathy.


Assuntos
Cardiomiopatia Hipertrófica/genética , Contração Miocárdica , Miosinas Ventriculares/genética , Animais , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/fisiopatologia , Humanos , Mutação , Miosinas Ventriculares/metabolismo
3.
Cardiovasc Diabetol ; 18(1): 13, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30696455

RESUMO

BACKGROUND: The diabetic heart undergoes remodelling contributing to an increased incidence of heart failure in individuals with diabetes at a later stage. The molecular regulators that drive this process in the diabetic heart are still unknown. METHODS: Real-time (RT) PCR analysis was performed to determine the expression of cardiac specific microRNA-208a in right atrial appendage (RAA) and left ventricular (LV) biopsy tissues collected from diabetic and non-diabetic patients undergoing coronary artery bypass graft surgery. To determine the time-dependent changes, cardiac tissue were collected from type 2 diabetic mice at different age groups. A western blotting analysis was conducted to determine the expression of contractile proteins α- and ß-myosin heavy chain (MHC) and thyroid hormone receptor-α (TR-α), the negative regulator of ß-MHC. To determine the beneficial effects of therapeutic modulation of miR-208a, high glucose treated adult mouse HL-1 cardiomyocytes were transfected with anti-miR-208a. RESULTS: RT-PCR analysis showed marked upregulation of miR-208a from early stages of diabetes in type 2 diabetic mouse heart, which was associated with a marked increase in the expression of pro-hypertrophic ß-MHC and downregulation of TR-α. Interestingly, upregulation of miR-208a preceded the switch of α-/ß-MHC isoforms and the development of diastolic and systolic dysfunction. We also observed significant upregulation of miR-208a and modulation of miR-208a associated proteins in the type 2 human diabetic heart. Therapeutic inhibition of miR-208a activity in high glucose treated HL-1 cardiomyocytes prevented the activation of ß-MHC and hence the hypertrophic response. CONCLUSION: Our results provide the first evidence that early modulation of miR-208a in the diabetic heart induces alterations in the downstream signaling pathway leading to cardiac remodelling and that therapeutic inhibition of miR-208a may be beneficial in preventing diabetes-induced adverse remodelling of the heart.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Ventrículos do Coração/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , MicroRNAs/metabolismo , Função Ventricular Esquerda , Remodelação Ventricular , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/genética , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Ventrículos do Coração/fisiopatologia , Humanos , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Pessoa de Meia-Idade , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Transdução de Sinais , Fatores de Tempo , Miosinas Ventriculares/genética , Miosinas Ventriculares/metabolismo
4.
Circ Res ; 123(6): 686-699, 2018 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-30355234

RESUMO

RATIONALE: Regeneration of lost cardiomyocytes is a fundamental unresolved problem leading to heart failure. Despite several strategies developed from intensive studies performed in the past decades, endogenous regeneration of heart tissue is still limited and presents a big challenge that needs to be overcome to serve as a successful therapeutic option for myocardial infarction. OBJECTIVE: One of the essential prerequisites for cardiac regeneration is the identification of endogenous cardiomyocyte progenitors and their niche that can be targeted by new therapeutic approaches. In this context, we hypothesized that the vascular wall, which was shown to harbor different types of stem and progenitor cells, might serve as a source for cardiac progenitors. METHODS AND RESULTS: We describe generation of spontaneously beating mouse aortic wall-derived cardiomyocytes without any genetic manipulation. Using aortic wall-derived cells (AoCs) of WT (wild type), αMHC (α-myosin heavy chain), and Flk1 (fetal liver kinase 1)-reporter mice and magnetic bead-associated cell sorting sorting of Flk1+ AoCs from GFP (green fluorescent protein) mice, we identified Flk1+CD (cluster of differentiation) 34+Sca-1 (stem cell antigen-1)-CD44- AoCs as the population that gives rise to aortic wall-derived cardiomyocytes. This AoC subpopulation delivered also endothelial cells and macrophages with a particular accumulation within the aortic wall-derived cardiomyocyte containing colonies. In vivo, cardiomyocyte differentiation capacity was studied by implantation of fluorescently labeled AoCs into chick embryonic heart. These cells acquired cardiomyocyte-like phenotype as shown by αSRA (α-sarcomeric actinin) expression. Furthermore, coronary adventitial Flk1+ and CD34+ cells proliferated, migrated into the myocardium after mouse myocardial infarction, and expressed Isl-1+ (insulin gene enhancer protein-1) indicative of cardiovascular progenitor potential. CONCLUSIONS: Our data suggest Flk1+CD34+ vascular adventitia-resident stem cells, including those of coronary adventitia, as a novel endogenous source for generating cardiomyocytes. This process is essentially supported by endothelial cells and macrophages. In summary, the therapeutic manipulation of coronary adventitia-resident cardiac stem and their supportive cells may open new avenues for promoting cardiac regeneration and repair after myocardial infarction and for preventing heart failure.


Assuntos
Túnica Adventícia/citologia , Aorta Torácica/citologia , Diferenciação Celular , Proliferação de Células , Miócitos Cardíacos/fisiologia , Células-Tronco/fisiologia , Animais , Antígenos CD34/metabolismo , Antígenos Ly/metabolismo , Células Cultivadas , Embrião de Galinha , Modelos Animais de Doenças , Feminino , Genes Reporter , Separação Imunomagnética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/cirurgia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/transplante , Cadeias Pesadas de Miosina/genética , Fenótipo , Regeneração , Transplante de Células-Tronco , Células-Tronco/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Miosinas Ventriculares/genética
5.
Proc Natl Acad Sci U S A ; 115(35): E8143-E8152, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30104387

RESUMO

Mutations in ß-cardiac myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. However, measurements of the intrinsic force, velocity, and ATPase activity of myosin have not provided a consistent mechanism to link mutations to muscle pathology. An alternative model posits that mutations in myosin affect the stability of a sequestered, super relaxed state (SRX) of the protein with very slow ATP hydrolysis and thereby change the number of myosin heads accessible to actin. Here we show that purified human ß-cardiac myosin exists partly in an SRX and may in part correspond to a folded-back conformation of myosin heads observed in muscle fibers around the thick filament backbone. Mutations that cause hypertrophic cardiomyopathy destabilize this state, while the small molecule mavacamten promotes it. These findings provide a biochemical and structural link between the genetics and physiology of cardiomyopathy with implications for therapeutic strategies.


Assuntos
Benzilaminas/química , Uracila/análogos & derivados , Miosinas Ventriculares/química , Animais , Benzilaminas/farmacologia , Cardiomegalia/enzimologia , Cardiomegalia/genética , Humanos , Músculo Esquelético/enzimologia , Mutação , Suínos , Porco Miniatura , Uracila/química , Uracila/farmacologia , Miosinas Ventriculares/genética , Miosinas Ventriculares/metabolismo
6.
Nat Struct Mol Biol ; 25(6): 505-514, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29867217

RESUMO

Concepts in molecular tension sensing in biology are growing and have their origins in studies of muscle contraction. In the heart muscle, a key parameter of contractility is the detachment rate of myosin from actin, which determines the time that myosin is bound to actin in a force-producing state and, importantly, depends on the load (force) against which myosin works. Here we measure the detachment rate of single molecules of human ß-cardiac myosin and its load dependence. We find that both can be modulated by both small-molecule compounds and cardiomyopathy-causing mutations. Furthermore, effects of mutations can be reversed by introducing appropriate compounds. Our results suggest that activating versus inhibitory perturbations of cardiac myosin are discriminated by the aggregate result on duty ratio, average force, and ultimately average power output and suggest that cardiac contractility can be controlled by tuning the load-dependent kinetics of single myosin molecules.


Assuntos
Miosinas Ventriculares/metabolismo , Actinas/metabolismo , Animais , Bovinos , Relação Dose-Resposta a Droga , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Humanos , Cinética , Mutação , Contração Miocárdica , Ureia/administração & dosagem , Ureia/análogos & derivados , Ureia/farmacologia , Miosinas Ventriculares/genética
7.
Open Biol ; 8(4)2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29669825

RESUMO

Myosin transduces ATP free energy into mechanical work in muscle. Cardiac muscle has dynamically wide-ranging power demands on the motor as the muscle changes modes in a heartbeat from relaxation, via auxotonic shortening, to isometric contraction. The cardiac power output modulation mechanism is explored in vitro by assessing single cardiac myosin step-size selection versus load. Transgenic mice express human ventricular essential light chain (ELC) in wild- type (WT), or hypertrophic cardiomyopathy-linked mutant forms, A57G or E143K, in a background of mouse α-cardiac myosin heavy chain. Ensemble motility and single myosin mechanical characteristics are consistent with an A57G that impairs ELC N-terminus actin binding and an E143K that impairs lever-arm stability, while both species down-shift average step-size with increasing load. Cardiac myosin in vivo down-shifts velocity/force ratio with increasing load by changed unitary step-size selections. Here, the loaded in vitro single myosin assay indicates quantitative complementarity with the in vivo mechanism. Both have two embedded regulatory transitions, one inhibiting ADP release and a second novel mechanism inhibiting actin detachment via strain on the actin-bound ELC N-terminus. Competing regulators filter unitary step-size selection to control force-velocity modulation without myosin integration into muscle. Cardiac myosin is muscle in a molecule.


Assuntos
Miosinas Cardíacas/fisiologia , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/fisiologia , Animais , Miosinas Cardíacas/química , Miosinas Cardíacas/genética , Humanos , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/genética , Miosinas Ventriculares/química , Miosinas Ventriculares/genética , Miosinas Ventriculares/fisiologia
8.
J Mol Cell Cardiol ; 119: 19-27, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29654880

RESUMO

The cardiac muscle sarcomere contains multiple proteins contributing to contraction energy transduction and its regulation during a heartbeat. Inheritable heart disease mutants affect most of them but none more frequently than the ventricular myosin motor and cardiac myosin binding protein c (mybpc3). These co-localizing proteins have mybpc3 playing a regulatory role to the energy transducing motor. Residue substitution and functional domain assignment of each mutation in the protein sequence decides, under the direction of a sensible disease model, phenotype and pathogenicity. The unknown model mechanism is decided here using a method combing neural and Bayes networks. Missense single nucleotide polymorphisms (SNPs) are clues for the disease mechanism summarized in an extensive database collecting mutant sequence location and residue substitution as independent variables that imply the dependent disease phenotype and pathogenicity characteristics in 4 dimensional data points (4ddps). The SNP database contains entries with the majority having one or both dependent data entries unfulfilled. A neural network relating causes (mutant residue location and substitution) and effects (phenotype and pathogenicity) is trained, validated, and optimized using fulfilled 4ddps. It then predicts unfulfilled 4ddps providing the implicit disease model. A discrete Bayes network interprets fulfilled and predicted 4ddps with conditional probabilities for phenotype and pathogenicity given mutation location and residue substitution thus relating the neural network implicit model to explicit features of the motor and mybpc3 sequence and structural domains. Neural/Bayes network forecasting automates disease mechanism modeling by leveraging the world wide human missense SNP database that is in place and expanding.


Assuntos
Proteínas de Transporte/genética , Cardiopatias/genética , Proteínas Musculares/genética , Contração Miocárdica/genética , Teorema de Bayes , Cardiopatias/diagnóstico , Cardiopatias/fisiopatologia , Humanos , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Miocárdio/patologia , Redes Neurais de Computação , Polimorfismo de Nucleotídeo Único/genética , Sarcômeros/genética , Miosinas Ventriculares/genética
9.
Dev Cell ; 44(1): 87-96.e5, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29316444

RESUMO

Truncating mutations in the sarcomere protein titin cause dilated cardiomyopathy due to sarcomere insufficiency. However, it remains mechanistically unclear how these mutations decrease sarcomere content in cardiomyocytes. Utilizing human induced pluripotent stem cell-derived cardiomyocytes, CRISPR/Cas9, and live microscopy, we characterize the fundamental mechanisms of human cardiac sarcomere formation. We observe that sarcomerogenesis initiates at protocostameres, sites of cell-extracellular matrix adhesion, where nucleation and centripetal assembly of α-actinin-2-containing fibers provide a template for the fusion of Z-disk precursors, Z bodies, and subsequent striation. We identify that ß-cardiac myosin-titin-protocostamere form an essential mechanical connection that transmits forces required to direct α-actinin-2 centripetal fiber assembly and sarcomere formation. Titin propagates diastolic traction stresses from ß-cardiac myosin, but not α-cardiac myosin or non-muscle myosin II, to protocostameres during sarcomerogenesis. Ablating protocostameres or decoupling titin from protocostameres abolishes sarcomere assembly. Together these results identify the mechanical and molecular components critical for human cardiac sarcomerogenesis.


Assuntos
Actinina/metabolismo , Junções Célula-Matriz/fisiologia , Conectina/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Sarcômeros/fisiologia , Miosinas Ventriculares/metabolismo , Actinina/genética , Adolescente , Adulto , Células Cultivadas , Conectina/genética , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/fisiologia , Miosinas Ventriculares/genética
10.
Gene ; 642: 335-342, 2018 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-29158167

RESUMO

Isoprenaline, an activator of ß-adrenergic receptor, has been found to induce cardiac hypertrophy in vivo and in vitro, but the exact mechanism is still unclear. ClC-3 is a member of the chloride channel family and is highly expressed in mammalian myocardium. In the present study, the role of ClC-3 in isopronaline-induced cardiac hypertrophy was investigated. We found that ClC-3 expression was reduced in isoprenaline-induced hypertrophic H9c2 cells, primary rat neonatal cardiomyocytes and myocardium of C57/BL/6 mice, and this reduction was prevented by the pretreatment of propranolol. Adeno-associated virus 9 (AAV9)-mediated ClC-3 expression in myocardium decreased heart mass index, thinned interventricular septum and left ventricular wall and lowered the mRNA expression of natriuretic peptide type A (ANF) and ß-myosin heavy chain (ß-MHC). Our results showed that ClC-3 played an important role in ß-adrenergic cardiac hypertrophy which could be associated with ANF and ß-MHC, and all these findings suggested that ClC-3 may be a novel therapeutic target for the prevention or treatment of myocardiac hypertrophy.


Assuntos
Cardiomegalia/metabolismo , Canais de Cloreto/metabolismo , Isoproterenol/efeitos adversos , Miócitos Cardíacos/metabolismo , Animais , Animais Recém-Nascidos , Cardiomegalia/induzido quimicamente , Cardiomegalia/genética , Cardiomegalia/prevenção & controle , Linhagem Celular , Canais de Cloreto/genética , Dependovirus/genética , Modelos Animais de Doenças , Regulação para Baixo , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Propranolol/farmacologia , Ratos , Receptores do Fator Natriurético Atrial/genética , Miosinas Ventriculares/genética
11.
Mol Med Rep ; 17(3): 3912-3920, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29257325

RESUMO

Ischemic heart disease within developed countries has been associated with high rates of morbidity and mortality. Cell­based cardiac repair is an emerging therapy for the treatment of cardiac diseases; however, a limited source of the optimal type of donor cell, such as an autologous cardiomyocyte, restricts clinical application. The novel therapeutic use of induced pluripotent stem cells (iPSCs) may serve as a unique and unlimited source of cardiomyocytes; however, iPSC contamination has been associated with teratoma formation following transplantation. The present study investigated whether cardiomyocytes from mouse fibroblasts may be reprogrammed in vitro with four cardiac transcription factors, including GATA binding protein 4, myocyte­specific enhancer factor 2C, T­box transcription factor 5, and heart­ and neural crest derivatives­expressed protein 2 (GMTH). Cardiac­specific markers, including α­myosin heavy chain (α­MHC), ß­MHC, atrial natriuretic factor, NK2 homeobox 5 and cardiac troponin T were observed within mouse fibroblasts reprogrammed with GMTH, which was reported to be more effective than GMT. In addition, Percoll density centrifugation enriched a population of ~72.4±5.5% α­MHC+ induced cardiomyocytes, which retained the expression profile of cardiomyocyte markers and were similar to natural neonatal cardiomyocytes in well­defined sarcomeric structures. The findings of the present study provided a potential solution to myocardial repair via a cell therapy applying tissue engineering with minimized risks of immune rejection and tumor formation.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Reprogramação Celular , Fibroblastos/metabolismo , Fator de Transcrição GATA4/genética , Fatores de Transcrição MEF2/genética , Miócitos Cardíacos/metabolismo , Proteínas com Domínio T/genética , Animais , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/metabolismo , Fibroblastos/citologia , Fator de Transcrição GATA4/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Fatores de Transcrição MEF2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Miócitos Cardíacos/citologia , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Cultura Primária de Células , Proteínas com Domínio T/metabolismo , Cauda/citologia , Cauda/metabolismo , Transdução Genética/métodos , Troponina T/genética , Troponina T/metabolismo , Miosinas Ventriculares/genética , Miosinas Ventriculares/metabolismo
12.
J Struct Biol ; 200(3): 219-228, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28743637

RESUMO

X-ray structural determination of segments of the myosin rod has proved difficult because of the strong salt-dependent aggregation properties and repeating pattern of charges on the surface of the coiled-coil that lead to the formation of paracrystals. This problem has been resolved in part through the use of globular assembly domains that improve protein folding and prevent aggregation. The primary consideration now in designing coiled-coil fusion constructs for myosin is deciding where to truncate the coiled-coil and which amino acid residues to include from the folding domain. This is especially important for myosin that contains numerous regions of low predicted coiled-coil propensity. Here we describe the strategy adopted to determine the structure of the region that extends from Arg1677 - Leu1797 that included two areas that do not show a strong sequence signature of a conventional left-handed coiled coil or canonical heptad repeat. This demonstrates again that, with careful choice of fusion constructs, overlapping structures exhibit very similar conformations for the myosin rod fragments in the canonical regions. However, conformational variability is seen around Leu1706 which is a hot spot for cardiomyopathy mutations suggesting that this might be important for function.


Assuntos
Subfragmentos de Miosina/química , Miosinas Ventriculares/química , Cristalografia por Raios X , Humanos , Leucina/genética , Modelos Moleculares , Subfragmentos de Miosina/genética , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Miosinas Ventriculares/genética
13.
Cell Physiol Biochem ; 42(4): 1645-1656, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28746924

RESUMO

BACKGROUND/AIMS: Adult cardiomyocytes can re-enter cell cycle as stimulated by prohypertrophic factors although they withdraw from cell cycle soon after birth. p21WAF1/CIP1, a cyclin-dependent kinase inhibitor, has been implicated in cardiac hypertrophy, however, its precise contribution to this process remains largely unclear. METHODS: The gene expression profile in left ventricle (LV) of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats was determined using quantitative PCR array and verified by real-time PCR and Western blotting. Hypertrophic response of H9c2 cells and neonatal rat ventricular myocytes (NRVM) were induced by angiotensin II (1 µmol/L). Cardiac hypertrophy of mice was elicited by isoproterenol (ISO) infusion (40 mg/kg per day for 14 days). p21-adenovirus and p21-siRNA were employed to transfect NRVM, and sterigmatocystin (STE, 3 mg/kg, ip, qd) was used to inhibit p21 activity. mRNA and protein expression levels of α- and ß-myosin heavy chain (MHC), p21WAF1/CIP1, calcineurin (CaN) and atrial natriuretic peptide (ANP) were assayed by realtime PCR and WB, respectively. RESULTS: Sixteen genes showed two-fold or greater changes between SHR and WKY rats, in which the expression of p21WAF1/CIP1 was upregulated by 4.15-fold (P=0.002) and reversed by losartan. Surface area, protein content, mRNA and protein expressions of ß-MHC, ANP and p21WAF1/CIP1 in H9c2 cells treated with AngII elevated significantly compared with control group. p21-Ad transfection markedly increased the surface area and ß-MHC mRNA expression of normal NRVMs, and p21-siRNA transfection decreased them in AngII-treated NRVMs. STE treatment decreased HW/BW and cross-sectional area, expression levels of ß-MHC, ANP and p21 significantly in ISO-treated mice. CONCLUSION: Our findings suggest that p21 facilitates the development of cardiac hypertrophy, and regulating the expression of p21 may be an approach to attenuate hypertrophic growth of cardiomyocytes.


Assuntos
Cardiomegalia/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Calcineurina/genética , Calcineurina/metabolismo , Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Isoproterenol , Losartan/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Esterigmatocistina/farmacologia , Miosinas Ventriculares/genética , Miosinas Ventriculares/metabolismo
14.
Arch Biochem Biophys ; 629: 19-35, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28688768

RESUMO

After acute myocardial infarction (AMI), neutrophils are recruited to the affected myocardium. Hypochlorous acid (HOCl) produced by neutrophil myeloperoxidase (MPO) damages cardiomyocytes and potentially expands the primary infarct. Rat cardiomyocyte-like cells were incubated with isolated human neutrophils treated with chemical activators in the absence or presence of nitroxide 4-methoxy-Tempo (MetT; 25 µM) for 4, 6 or 24 h; studies with reagent HOCl served as positive control. Treating cardiomyocytes with activated neutrophils or reagent HOCl resulted in a marked increase in protein tyrosine chlorination and a decline in protein tyrosine phosphatase (PTP) activity. On balance our data also supported an increase in phosphorylation of MAPK p38 and ERK1/2 suggestive of an intracellular hyperphosphorylation status and this was accompanied by decreases in cell viability, as judged by assessing caspases-3/7 activity. For cells exposed to activated neutrophils receptor-mediated uptake of transferrin decreased although total matrix metalloproteinase (MMP) activity was unaffected. Addition of MetT ameliorated protein tyrosine chlorination, decreased MAPK activity and restored receptor-mediated transferrin uptake and PTP activity in cardiomyocytes. Overall, adverse effects of neutrophil-derived HOCl on cultured cardiomyocytes were ameliorated by MetT suggesting that nitroxides may be beneficial to inflammatory pathologies, where neutrophil recruitment/activation is a prominent and early feature.


Assuntos
Óxidos N-Cíclicos/farmacologia , Neutrófilos/metabolismo , Proteínas Quinases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Neutrófilos/enzimologia , Especificidade de Órgãos , Estresse Oxidativo/efeitos dos fármacos , Peroxidase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ratos , Transferrina/metabolismo , Tirosina/metabolismo , Miosinas Ventriculares/genética
15.
Cell Mol Biol (Noisy-le-grand) ; 63(4): 23-27, 2017 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-28478799

RESUMO

Cardiac hypertrophy is a crucial predictor of heart failure and is regulated by microRNAs. MicroRNA-124 (miR-124) is regarded as a prognostic indicator for outcomes after cardiac arrest. However, whether miR-124 participates in cardiac hypertrophy remains unclear. Therefore, our study aimed to determine the role of miR-124 in angiotensin II(AngII)-induced myocardial hypertrophy and the possible mechanism. Primary cultured rat neonatal cardiomyocytes(NCMs) were transfected with miR-124 mimics or inhibitor, followed by AngII stimulation. Quantitative RT-PCR, western blot analysis and determination of cell surface area of NCMs were used to detect the hypertrophic phenotypes. We observed that miR-124 was elevated in AngII-induced hypertrophic cardiomyocytes. Cell surface area of NCMs and mRNA expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and ß-myosin heavy chain (ß-MHC), indicators of myocardial hypertrophy, were higher in NCMs transfected with miR-124 mimics in the presence of AngII. On the contrary, knockdown of miR-124 by its specific inhibitor could restore these courses. Furthermore, downregulation of miR-124 alleviated the increased protein level of endoplasmic reticulum (ER) stress markers 78-kDa glucose-regulated protein (Grp78) and calreticulin(CRT) in AngII-induced NCMs. In conclusion, our study shows that inhibition of miR-124 effectively suppresses AngII-induced myocardial hypertrophy, which is associated with attenuation of ER stress.


Assuntos
Angiotensina II/administração & dosagem , Cardiomegalia/genética , Estresse do Retículo Endoplasmático/genética , MicroRNAs/genética , Angiotensina II/metabolismo , Animais , Fator Natriurético Atrial/genética , Calreticulina/genética , Cardiomegalia/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Humanos , MicroRNAs/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Peptídeo Natriurético Encefálico/genética , Ratos , Miosinas Ventriculares/genética
16.
Cardiovasc Res ; 113(10): 1124-1136, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28371863

RESUMO

Aims: The E143K (Glu → Lys) mutation in the myosin essential light chain has been associated with restrictive cardiomyopathy (RCM) in humans, but the mechanisms that underlie the development of defective cardiac function are unknown. Using transgenic E143K-RCM mice, we sought to determine the molecular and cellular triggers of E143K-induced heart remodelling. Methods and results: The E143K-induced abnormalities in cardiac function and morphology observed by echocardiography and invasive haemodynamics were paralleled by augmented active and passive tension measured in skinned papillary muscle fibres compared with wild-type (WT)-generated force. In vitro, E143K-myosin had increased duty ratio and binding affinity to actin compared with WT-myosin, increased actin-activated ATPase activity and slower rates of ATP-dependent dissociation of the acto-myosin complex, indicating an E143K-induced myosin hypercontractility. E143K was also observed to reduce the level of myosin regulatory light chain phosphorylation while that of troponin-I remained unchanged. Small-angle X-ray diffraction data showed a decrease in the filament lattice spacing (d1,0) with no changes in the equatorial reflections intensity ratios (I1,1/I1,0) in E143K vs. WT skinned papillary muscles. The hearts of mutant-mice demonstrated ultrastructural defects and fibrosis that progressively worsened in senescent animals and these changes were hypothesized to contribute to diastolic disturbance and to mild systolic dysfunction. Gene expression profiles of E143K-hearts supported the histopathology results and showed an upregulation of stress-response and collagen genes. Finally, proteomic analysis evidenced RCM-dependent metabolic adaptations and higher energy demands in E143K vs. WT hearts. Conclusions: As a result of the E143K-induced myosin hypercontractility, the hearts of RCM mice model exhibited cardiac dysfunction, stiff ventricles and physiological, morphologic, and metabolic remodelling consistent with the development of RCM. Future efforts should be directed toward normalization of myosin motor function and the use of myosin-specific therapeutics to avert the hypercontractile state of E143K-myosin and prevent pathological cardiac remodelling.


Assuntos
Cardiomiopatia Restritiva/genética , Mutação , Contração Miocárdica/genética , Miócitos Cardíacos/patologia , Cadeias Leves de Miosina/genética , Sarcômeros/patologia , Função Ventricular Esquerda/genética , Miosinas Ventriculares/genética , Remodelação Ventricular/genética , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cardiomiopatia Restritiva/metabolismo , Cardiomiopatia Restritiva/patologia , Cardiomiopatia Restritiva/fisiopatologia , Colágeno/metabolismo , Modelos Animais de Doenças , Metabolismo Energético , Feminino , Fibrose , Predisposição Genética para Doença , Humanos , Masculino , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Cadeias Leves de Miosina/metabolismo , Fenótipo , Fosforilação , Sarcômeros/metabolismo , Sarcômeros/ultraestrutura , Miosinas Ventriculares/metabolismo
17.
Cardiovasc Res ; 113(8): 915-925, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28379313

RESUMO

Background: Dilated cardiomoypathies (DCM) are a heterogeneous group of inherited and acquired diseases characterized by decreased contractility and enlargement of cardiac chambers and a major cause of morbidity and mortality. Mice with Glu54Lys mutation in α-tropomyosin (Tm54) demonstrate typical DCM phenotype with reduced myofilament Ca2+ sensitivity. We tested the hypothesis that early sensitization of the myofilaments to Ca2+ in DCM can prevent the DCM phenotype. Methods and results: To sensitize Tm54 myofilaments, we used a genetic approach and crossbred Tm54 mice with mice expressing slow skeletal troponin I (ssTnI) that sensitizes myofilaments to Ca2+. Four groups of mice were used: non-transgenic (NTG), Tm54, ssTnI and Tm54/ssTnI (DTG). Systolic function was significantly reduced in the Tm54 mice compared to NTG, but restored in DTG mice. Tm54 mice also showed increased diastolic LV dimensions and HW/BW ratios, when compared to NTG, which were improved in the DTG group. ß-myosin heavy chain expression was increased in the Tm54 animals compared to NTG and was partially restored in DTG group. Analysis by 2D-DIGE indicated a significant decrease in two phosphorylated spots of cardiac troponin I (cTnI) in the DTG animals compared to NTG and Tm54. Analysis by 2D-DIGE also indicated no significant changes in troponin T, regulatory light chain, myosin binding protein C and tropomyosin phosphorylation. Conclusion: Our data indicate that decreased myofilament Ca2+ sensitivity is an essential element in the pathophysiology of thin filament linked DCM. Sensitization of myofilaments to Ca2+ in the early stage of DCM may be a useful therapeutic strategy in thin filament linked DCM.


Assuntos
Citoesqueleto de Actina/metabolismo , Cardiomiopatia Dilatada/genética , Animais , Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Camundongos Transgênicos , Miocárdio/metabolismo , Fosforilação , Tropomiosina/metabolismo , Miosinas Ventriculares/genética
18.
Sci Adv ; 3(2): e1601959, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28246639

RESUMO

Hypertrophic cardiomyopathy (HCM) affects 1 in 500 individuals and is an important cause of arrhythmias and heart failure. Clinically, HCM is characterized as causing hypercontractility, and therapies are aimed toward controlling the hyperactive physiology. Mutations in the ß-cardiac myosin comprise ~40% of genetic mutations associated with HCM, and the converter domain of myosin is a hotspot for HCM-causing mutations; however, the underlying primary effects of these mutations on myosin's biomechanical function remain elusive. We hypothesize that these mutations affect the biomechanical properties of myosin, such as increasing its intrinsic force and/or its duty ratio and therefore the ensemble force of the sarcomere. Using recombinant human ß-cardiac myosin, we characterize the molecular effects of three severe HCM-causing converter domain mutations: R719W, R723G, and G741R. Contrary to our hypothesis, the intrinsic forces of R719W and R723G mutant myosins are decreased compared to wild type and unchanged for G741R. Actin and regulated thin filament gliding velocities are ~15% faster for R719W and R723G myosins, whereas there is no change in velocity for G741R. Adenosine triphosphatase activities and the load-dependent velocity change profiles of all three mutant proteins are very similar to those of wild type. These results indicate that the net biomechanical properties of human ß-cardiac myosin carrying these converter domain mutations are very similar to those of wild type or are even slightly hypocontractile, leading us to consider an alternative mechanism for the clinically observed hypercontractility. Future work includes how these mutations affect protein interactions within the sarcomere that increase the availability of myosin heads participating in force production.


Assuntos
Cardiomegalia , Doenças Genéticas Inatas , Miosinas Ventriculares/química , Substituição de Aminoácidos , Humanos , Mutação de Sentido Incorreto , Miosinas Ventriculares/genética , Miosinas Ventriculares/metabolismo
19.
J Mol Cell Cardiol ; 103: 93-101, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28089740

RESUMO

Familial hypertrophic cardiomyopathy (HCM), linked to mutations in myosin, myosin-binding proteins and other sarcolemmal proteins, is associated with increased risk of life threatening ventricular arrhythmias, and a number of animal models have been developed to facilitate analysis of disease progression and mechanisms. In the experiments here, we use the αMHC403/+ mouse line in which one αMHC allele harbors a common HCM mutation (in ßMHC, Arg403 Gln). Here, we demonstrate marked prolongation of QT intervals in young adult (10-12week) male αMHC403/+ mice, well in advance of the onset of measurable left ventricular hypertrophy. Electrophysiological recordings from myocytes isolated from the interventricular septum of these animals revealed significantly (P<0.001) lower peak repolarizing voltage-gated K+ (Kv) current (IK,peak) amplitudes, compared with cells isolated from wild type (WT) littermate controls. Analysis of Kv current waveforms revealed that the amplitudes of the inactivating components of the total outward Kv current, Ito,f, Ito,s and IK,slow, were significantly lower in αMHC403/+, compared with WT, septum cells, whereas Iss amplitudes were similar. The amplitudes/densities of IK,peak and IK,slow were also lower in αMHC403/+, compared with WT, LV wall and LV apex myocytes, whereas Ito,f was attenuated in αMHC403/+ LV wall, but not LV apex, cells. These regional differences in the remodeling of repolarizing Kv currents in the αMHC403/+ mice would be expected to increase the dispersion of ventricular repolarization and be proarrhythmic. Quantitative RT-PCR analysis revealed reductions in the expression of transcripts encoding several K+ channel subunits in the interventricular septum, LV free wall and LV apex of (10-12week) αMHC403/+ mice, although this transcriptional remodeling was not correlated with the observed decreases in K+ current amplitudes.


Assuntos
Potenciais de Ação , Cardiomiopatia Hipertrófica Familiar/etiologia , Cardiomiopatia Hipertrófica Familiar/metabolismo , Mutação , Miocárdio/metabolismo , Canais de Potássio/metabolismo , Miosinas Ventriculares/genética , Animais , Biópsia , Cardiomiopatia Hipertrófica Familiar/diagnóstico , Modelos Animais de Doenças , Ecocardiografia , Eletrocardiografia , Expressão Gênica , Perfilação da Expressão Gênica , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Camundongos , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Remodelação Ventricular/genética
20.
J Muscle Res Cell Motil ; 37(6): 215-223, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27975185

RESUMO

The N-terminal extension of human cardiac troponin T (TnT), which modulates myofilament Ca2+ sensitivity, contains several hypertrophic cardiomyopathy (HCM)-causing mutations including S69F. However, the functional consequence of S69F mutation is unknown. The human analog of S69F in rat TnT is L71F (TnTL71F). Because the functional consequences due to structural changes in the N-terminal extension are influenced by the type of myosin heavy chain (MHC) isoform, we hypothesized that the TnTL71F-mediated effect would be differently modulated by α- and ß-MHC isoforms. TnTL71F and wild-type rat TnT were reconstituted into de-membranated muscle fibers from normal (α-MHC) and propylthiouracil-treated rat hearts (ß-MHC) to measure steady-state and dynamic contractile parameters. The magnitude of the TnTL71F-mediated attenuation of Ca2+-activated maximal tension was greater in α- than in ß-MHC fibers. For example, TnTL71F attenuated maximal tension by 31% in α-MHC fibers but only by 10% in ß-MHC fibers. Furthermore, TnTL71F reduced myofilament Ca2+ sensitivity by 0.11 pCa units in α-MHC fibers but only by 0.05 pCa units in ß-MHC fibers. TnTL71F augmented rate constants of crossbridge recruitment and crossbridge detachment dynamics in α-MHC fibers but not in ß-MHC fibers. Collectively, our data demonstrate that TnTL71F induces greater contractile deficits against α-MHC than against ß-MHC background.


Assuntos
Mutação/genética , Cadeias Pesadas de Miosina/genética , Troponina T/genética , Miosinas Ventriculares/genética , Animais , Cálcio/metabolismo , Masculino , Contração Miocárdica/genética , Miofibrilas/genética , Isoformas de Proteínas/genética , Ratos , Ratos Sprague-Dawley
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