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1.
Adv Mater ; 35(17): e2207443, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36650030

RESUMO

A mechanistic understanding of cell-autonomous skeletal muscle changes after injury can lead to novel interventions to improve functional recovery in an aged population. However, major knowledge gaps persist owing to limitations of traditional biological aging models. 2D cell culture represents an artificial environment, while aging mammalian models are contaminated by influences from non-muscle cells and other organs. Here, a 3D muscle aging system is created to overcome the limitations of these traditional platforms. It is shown that old muscle constructs (OMC) manifest a sarcopenic phenotype, as evidenced by hypotrophic myotubes, reduced contractile function, and decreased regenerative capacity compared to young muscle constructs. OMC also phenocopy the regenerative responses of aged muscle to two interventions, pharmacological and biological. Interrogation of muscle cell-specific mechanisms that contribute to impaired regeneration over time further reveals that an aging-induced increase of complement component 4b (C4b) delays muscle progenitor cell amplification and impairs functional recovery. However, administration of complement factor I, a C4b inactivator, improves muscle regeneration in vitro and in vivo, indicating that C4b inhibition may be a novel approach to enhance aged muscle repair. Collectively, the model herein exhibits capabilities to study cell-autonomous changes in skeletal muscle during aging, regeneration, and intervention.


Assuntos
Complemento C4b , Músculo Esquelético , Animais , Envelhecimento/fisiologia , Fibras Musculares Esqueléticas , Contração Muscular , Mamíferos
2.
PLoS One ; 11(12): e0167681, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27936050

RESUMO

Most studies of the mechanisms leading to hereditary dilated cardiomyopathy (DCM) have been performed in reconstituted in vitro systems. Genetically engineered murine models offer the opportunity to dissect these mechanisms in vivo. We generated a gene-targeted knock-in murine model of the autosomal dominant Arg141Trp (R141W) mutation in Tnnt2, which was first described in a human family with DCM. Mice heterozygous for the mutation (Tnnt2R141W/+) recapitulated the human phenotype, developing left ventricular dilation and reduced contractility. There was a gene dosage effect, so that the phenotype in Tnnt2R141W/+mice was attenuated by transgenic overexpression of wildtype Tnnt2 mRNA transcript. Male mice exhibited poorer survival than females. Biomechanical studies on skinned fibers from Tnnt2R141W/+ hearts showed a significant decrease in pCa50 (-log[Ca2+] required for generation of 50% of maximal force) relative to wildtype hearts, indicating Ca2+ desensitization. Optical mapping studies of Langendorff-perfused Tnnt2R141W/+ hearts showed marked increases in diastolic and peak systolic intracellular Ca2+ ([Ca2+]i), and prolonged systolic rise and diastolic fall of [Ca2+]i. Perfused Tnnt2R141W/+ hearts had slower intrinsic rates in sinus rhythm and reduced peak heart rates in response to isoproterenol. Tnnt2R141W/+ hearts exhibited a reduction in phosphorylated phospholamban relative to wildtype mice. However, crossing Tnnt2R141W/+ mice with phospholamban knockout (Pln-/-) mice, which exhibit increased Ca2+ transients and contractility, had no effect on the DCM phenotype. We conclude that the Tnnt2 R141W mutation causes a Ca2+ desensitization and mice adapt by increasing Ca2+-transient amplitudes, which impairs Ca2+ handling dynamics, metabolism and responses to ß-adrenergic activation.


Assuntos
Cálcio/metabolismo , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Coração/fisiopatologia , Mutação Puntual , Troponina T/genética , Animais , Cardiomiopatia Dilatada/fisiopatologia , Feminino , Técnicas de Introdução de Genes , Marcação de Genes , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Contração Miocárdica
3.
Mar Pollut Bull ; 102(1): 187-98, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26621576

RESUMO

Fourteen surveys were conducted at Farallon De Medinilla (a U.S. Department of Defense bombing range in the Mariana Archipelago) between 1997 and 2012; annual surveys were conducted from 1999 through 2012. There was no evidence that the condition of the biological resources assessed had changed, or been adversely impacted to a significant degree by the training activities being conducted there. Restricted access has resulted in a de-facto preserve effect and outweighs minor negative impacts from training. The health, abundance and biomass of fishes, corals and other marine resources are comparable to or superior to those in similar habitats at other locations within the Mariana Archipelago. Our research suggests that the greatest threat to FDM's marine resources is from fishermen, not military training activities.


Assuntos
Conservação dos Recursos Naturais , Ecossistema , Monitoramento Ambiental , Animais , Antozoários , Biomassa , Pesqueiros , Peixes , Micronésia , Instalações Militares
4.
Circ Res ; 105(12): 1232-9, 2009 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-19850940

RESUMO

RATIONALE: Protein kinase (PK)C-induced phosphorylation of cardiac troponin (cTn)I has been shown to regulate cardiac contraction. OBJECTIVE: Characterize functional effects of increased PKC-induced cTnI phosphorylation and identify underlying mechanisms using a transgenic mouse model (cTnI(PKC-P)) expressing mutant cTnI (S43E, S45E, T144E). METHODS AND RESULTS: Two-dimensional gel analysis showed 7.2+/-0.5% replacement of endogenous cTnI with the mutant form. Experiments included: mechanical measurements (perfused isolated hearts, isolated papillary muscles, and skinned fiber preparations), biochemical and molecular biological measurements, and a mathematical model-based analysis for integrative interpretation. Compared to wild-type mice, cTnI(PKC-P) mice exhibited negative inotropy in isolated hearts (14% decrease in peak developed pressure), papillary muscles (53% decrease in maximum developed force), and skinned fibers (14% decrease in maximally activated force, F(max)). Additionally, cTnI(PKC-P) mice exhibited slowed relaxation in both isolated hearts and intact papillary muscles. The cTnI(PKC-P) mice showed no differences in calcium sensitivity, cooperativity, steady-state force-MgATPase relationship, calcium transient (amplitude and relaxation), or baseline phosphorylation of other myofilamental proteins. The model-based analysis revealed that experimental observations in cTnI(PKC-P) mice could be reproduced by 2 simultaneous perturbations: a decrease in the rate of cross-bridge formation and an increase in calcium-independent persistence of the myofilament active state. CONCLUSIONS: A modest increase in PKC-induced cTnI phosphorylation ( approximately 7%) can significantly alter cardiac muscle contraction: negative inotropy via decreased cross-bridge formation and negative lusitropy via persistence of myofilament active state. Based on our data and data from the literature we speculate that effects of PKC-mediated cTnI phosphorylation are site-specific (S43/S45 versus T144).


Assuntos
Contração Miocárdica , Miocárdio/enzimologia , Proteína Quinase C/metabolismo , Troponina I/metabolismo , Função Ventricular Esquerda , Citoesqueleto de Actina/enzimologia , Animais , Sinalização do Cálcio , Eletroforese em Gel Bidimensional , Camundongos , Camundongos Transgênicos , Modelos Cardiovasculares , Força Muscular , Mutação , Contração Miocárdica/genética , Músculos Papilares/enzimologia , Fosforilação , Troponina I/genética , Função Ventricular Esquerda/genética , Pressão Ventricular
5.
PLoS One ; 3(7): e2642, 2008 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-18612386

RESUMO

BACKGROUND: Hypertrophic (HCM) and dilated (DCM) cardiomyopathies result from sarcomeric protein mutations, including cardiac troponin T (cTnT, TNNT2). We determined whether TNNT2 mutations cause cardiomyopathies by altering cTnT function or quantity; whether the severity of DCM is related to the ratio of mutant to wildtype cTnT; whether Ca(2+) desensitization occurs in DCM; and whether absence of cTnT impairs early embryonic cardiogenesis. METHODS AND FINDINGS: We ablated Tnnt2 to produce heterozygous Tnnt2(+/-) mice, and crossbreeding produced homozygous null Tnnt2(-/-) embryos. We also generated transgenic mice overexpressing wildtype (TG(WT)) or DCM mutant (TG(K210Delta)) Tnnt2. Crossbreeding produced mice lacking one allele of Tnnt2, but carrying wildtype (Tnnt2(+/-)/TG(WT)) or mutant (Tnnt2(+/-)/TG(K210Delta)) transgenes. Tnnt2(+/-) mice relative to wildtype had significantly reduced transcript (0.82+/-0.06[SD] vs. 1.00+/-0.12 arbitrary units; p = 0.025), but not protein (1.01+/-0.20 vs. 1.00+/-0.13 arbitrary units; p = 0.44). Tnnt2(+/-) mice had normal hearts (histology, mass, left ventricular end diastolic diameter [LVEDD], fractional shortening [FS]). Moreover, whereas Tnnt2(+/-)/TG(K210Delta) mice had severe DCM, TG(K210Delta) mice had only mild DCM (FS 18+/-4 vs. 29+/-7%; p<0.01). The difference in severity of DCM may be attributable to a greater ratio of mutant to wildtype Tnnt2 transcript in Tnnt2(+/-)/TG(K210Delta) relative to TG(K210Delta) mice (2.42+/-0.08, p = 0.03). Tnnt2(+/-)/TG(K210Delta) muscle showed Ca(2+) desensitization (pCa(50) = 5.34+/-0.08 vs. 5.58+/-0.03 at sarcomere length 1.9 microm, p<0.01), but no difference in maximum force generation. Day 9.5 Tnnt2(-/-) embryos had normally looped hearts, but thin ventricular walls, large pericardial effusions, noncontractile hearts, and severely disorganized sarcomeres. CONCLUSIONS: Absence of one Tnnt2 allele leads to a mild deficit in transcript but not protein, leading to a normal cardiac phenotype. DCM results from abnormal function of a mutant protein, which is associated with myocyte Ca(2+) desensitization. The severity of DCM depends on the ratio of mutant to wildtype Tnnt2 transcript. cTnT is essential for sarcomere formation, but normal embryonic heart looping occurs without contractile activity.


Assuntos
Cardiomiopatia Dilatada/genética , Coração/embriologia , Troponina T/genética , Troponina T/fisiologia , Animais , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Hipertrófica/metabolismo , Ecocardiografia , Embrião de Mamíferos/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Miocárdio/metabolismo , Fenótipo , Troponina T/metabolismo
6.
J Biol Chem ; 283(15): 10135-46, 2008 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-18250163

RESUMO

Reversible acetylation of lysine residues within a protein is considered a biologically relevant modification that rivals phosphorylation ( Kouzarides, T. (2000) EMBO J. 19, 1176-1179 ). The enzymes responsible for such protein modification are called histone acetyltransferases (HATs) and deacetylases (HDACs). A role of protein phosphorylation in regulating muscle contraction is well established ( Solaro, R. J., Moir, A. J., and Perry, S. V. (1976) Nature 262, 615-617 ). Here we show that reversible protein acetylation carried out by HATs and HDACs also plays a role in regulating the myofilament contractile activity. We found that a Class II HDAC, HDAC4, and an HAT, PCAF, associate with cardiac myofilaments. Primary cultures of cardiomyocytes as well as mouse heart sections examined by immunohistochemical and electron microscopic analyses revealed that both HDAC4 and PCAF associate with the Z-disc and I- and A-bands of cardiac sarcomeres. Increased acetylation of sarcomeric proteins by HDAC inhibition (using class I and II HDAC inhibitors or anti-HDAC4 antibody) enhanced the myofilament calcium sensitivity. We identified the Z-disc-associated protein, MLP, a sensor of cardiac mechanical stretch, as an acetylated target of PCAF and HDAC4. We also show that trichostatin-A, a class I and II HDAC inhibitor, increases myofilament calcium sensitivity of wild-type, but not of MLP knock-out mice, thus demonstrating a role of MLP in acetylation-dependent increased contractile activity of myofilaments. These studies provide the first evidence that HATs and HDACs play a role in regulation of muscle contraction.


Assuntos
Histona Desacetilases/metabolismo , Contração Miocárdica/fisiologia , Miocárdio/enzimologia , Sarcômeros/enzimologia , Fatores de Transcrição de p300-CBP/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Cálcio/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Histona Desacetilases/genética , Ácidos Hidroxâmicos/farmacologia , Proteínas com Domínio LIM , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miocárdio/ultraestrutura , Miócitos Cardíacos , Coelhos , Ratos , Sarcômeros/genética , Sarcômeros/ultraestrutura , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Fatores de Transcrição de p300-CBP/genética
7.
J Mol Cell Cardiol ; 34(5): 547-54, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-12056858

RESUMO

Although ATP is the immediate source of energy for muscle contraction other nucleoside triphosphates (NTP) can substitute for ATP as substrates for myosin and as sources of energy for contraction of skinned muscle fibers. However, experiments with skinned skeletal muscle fibers in the presence of substitute NTP indicate significant differences with respect to cross-bridge kinetics, force generation, and Ca(2+) regulation. In this study the length dependence of Ca(2+) sensitivity of skinned bovine cardiac muscle was analyzed in the presence of MgATP, MgCTP, MgUTP, and MgITP. Ca(2+) regulation in the presence of MgCTP and MgUTP was essentially the same as in the presence of MgATP, although the maximum force generated (at sarcomere length 2.4 microm) was about 25% less. However, the length dependence of Ca(2+) sensitivity was eliminated in the presence of MgUTP. With MgITP the maximum force generated (at sarcomere length 2.4 microm) was about the same as in the presence of MgATP, but there was an impairment of relaxation such that at pCa 8 the force developed was about 50-60% of that developed at pCa 5. Moreover, the Ca(2+)-dependent component showed no length-dependent sensitivity. Thus length modulation of Ca(2+) sensitivity is a function of the myosin substrate. Taken in conjunction with other data, the results are consistent with the hypothesis that length-dependence of Ca(2+) sensitivity is modulated at a step upstream from the force-generating reaction.


Assuntos
Cálcio/fisiologia , Coração/fisiologia , Contração Miocárdica/fisiologia , Nucleotídeos/fisiologia , Sarcômeros/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Bovinos , Contração Miocárdica/efeitos dos fármacos , Miocárdio/citologia , Nucleotídeos/farmacologia
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