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1.
Adv Exp Med Biol ; 1441: 417-433, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38884723

RESUMO

This chapter will describe basic structural and functional features of the contractile apparatus of muscle cells of the heart, namely, cardiomyocytes and smooth muscle cells. Cardiomyocytes form the contractile myocardium of the heart, while smooth muscle cells form the contractile coronary vessels. Both muscle types have distinct properties and will be considered with respect to their cellular appearance (brick-like cross-striated versus spindle-like smooth), arrangement of contractile proteins (sarcomeric versus non-sarcomeric organization), calcium activation mechanisms (thin-filament versus thick-filament regulation), contractile features (fast and phasic versus slow and tonic), energy metabolism (high oxygen versus low oxygen demand), molecular motors (type II myosin isoenzymes with high adenosine diphosphate [ADP]-release rate versus myosin isoenzymes with low ADP-release rates), chemomechanical energy conversion (high adenosine triphosphate [ATP] consumption and short duty ratio versus low ATP consumption and high duty ratio of myosin II cross-bridges [XBs]), and excitation-contraction coupling (calcium-induced calcium release versus pharmacomechanical coupling). Part of the work has been published (Neuroscience - From Molecules to Behavior", Chap. 22, Galizia and Lledo eds 2013, Springer-Verlag; with kind permission from Springer Science + Business Media).


Assuntos
Contração Miocárdica , Miócitos Cardíacos , Humanos , Contração Miocárdica/fisiologia , Animais , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Cálcio/metabolismo , Metabolismo Energético , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Acoplamento Excitação-Contração/fisiologia
2.
Basic Res Cardiol ; 112(1): 1, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27837311

RESUMO

Chronic increased workload of the human heart causes ventricular hypertrophy, re-expression of the atrial essential myosin light chain (hALC-1), and improved contractile function. Although hALC-1 is an important positive inotropic regulator of the human heart, little is known about its regulation. Therefore, we investigated the role of the sex hormone 17ß-estradiol (E2) on hALC-1 gene expression, the underlying molecular mechanisms, and the impact of this regulatory process on cardiac contractile function. We showed that E2 attenuated hALC-1 expression in human atrial tissues of both sexes and in human ventricular AC16 cells. E2 induced the nuclear translocation of estrogen receptor alpha (ERα) and hALC-1 in AC16 cells, where they cooperatively regulate the transcriptional activity of hALC-1 gene promoter. E2-activated ERα required the estrogen response element (ERE) motif within the hALC-1 gene promoter to reduce its transcriptional activity (vehicle: 15.55 ± 4.80 vs. E2: 6.51 ± 3.69; ~2 fold). This inhibitory effect was potentiated in the presence of hALC-1 (vehicle: 11.13 ± 3.66 vs. E2: 2.18 ± 1.10; ~5 fold), and thus, hALC-1 acts as a co-repressor of ERα-mediated transcription. Yeast two-hybrid screening of a human heart cDNA library revealed that ERα interacts physically with hALC-1 in the presence of E2. This interaction was confirmed by Co-Immunoprecipitation and immunofluorescence in human atrium. As a further novel effect, we showed that chronic E2-treatment of adult mouse cardiomyocytes overexpressing hALC-1 resulted in reduced cell-shortening amplitude and twitching kinetics of these cells independent of Ca2+ activation levels. Together, our data showed that the expression of hALC-1 gene is, at least partly, regulated by E2/ERα, while hALC-1 acts as a co-repressor. The inotropic effect of hALC-1 overexpression in cardiomyocytes can be significantly repressed by E2.


Assuntos
Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Regulação da Expressão Gênica/genética , Contração Miocárdica/fisiologia , Cadeias Leves de Miosina/biossíntese , Animais , Western Blotting , Feminino , Imunofluorescência , Humanos , Imunoprecipitação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/genética , Reação em Cadeia da Polimerase , Técnicas do Sistema de Duplo-Híbrido
3.
J Vasc Res ; 54(3): 131-142, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28468000

RESUMO

AIM: Vascular remodeling following injury substantially accounts for restenosis and adverse clinical outcomes. In this study, we investigated the role of the giant scaffold protein Ahnak1 in vascular healing after endothelial denudation of the murine femoral artery. METHODS: The spatiotemporal expression pattern of Ahnak1 and Ahnak2 was examined using specific antibodies and real-time quantitative PCR. Following wire-mediated endothelial injury of Ahnak1-deficient mice and wild-type (WT) littermates, the processes of vascular healing were analyzed. RESULTS: Ahnak1 and Ahnak2 showed a mutually exclusive vascular expression pattern, with Ahnak1 being expressed in the endothelium and Ahnak2 in the medial cells in naïve WT arteries. After injury, a marked increase of Ahnak1- and Ahnak2-positive cells at the lesion site became evident. Both proteins showed a strong upregulation in neointimal cells 14 days after injury. Ahnak1-deficient mice showed delayed vascular healing and dramatically impaired re-endothelialization that resulted in prolonged adverse vascular remodeling, when compared to the WT littermates. CONCLUSION: The large scaffold and adaptor proteins Ahnak1 and Ahnak2 exhibit differential expression patterns and functions in naïve and injured arteries. Ahnak1 plays a nonredundant protective role in vascular healing.


Assuntos
Artéria Femoral/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Remodelação Vascular , Lesões do Sistema Vascular/metabolismo , Cicatrização , Animais , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/patologia , Artéria Femoral/lesões , Artéria Femoral/patologia , Genótipo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Neointima , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Fenótipo , Reepitelização , Fatores de Tempo , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/patologia
4.
Clin Sci (Lond) ; 130(5): 365-76, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26608078

RESUMO

ERß (oestrogen receptor ß) activation has been shown to be cardioprotective, but the cell types and mechanisms involved are not understood. To investigate whether ERß restricted to cardiomyocytes contributes to the observed cardioprotection, we tested the effects of cardiomyocyte-specific ERß-OE (ERß overexpression) on survival, cardiac remodelling and function after MI (myocardial infarction) and studied the molecular pathways potentially involved. Female and male mice with cardiomyocyte-specific ERß-OE and WT (wild-type) littermates were subjected to chronic anterior coronary artery ligation or sham surgery. Two weeks after MI, ERß-OE mice showed improved survival (100% and 83% compared with 76% and 58% in WT females and males respectively). ERß-OE was associated with attenuated LV (left ventricular) dilatation, smaller increase in heart weight, less lung congestion at similar MI size, and improved systolic and diastolic function in both sexes. We identified two potential pathways for ERß-mediated myocardial protection. First, male and female ERß-OE mice had a lower reduction of SERCA2a (sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 2a) expression after MI, suggesting less reduction in diastolic Ca(2+)-reuptake into the sarcoplasmic reticulum post-MI. Secondly, male ERß-OE revealed attenuated cardiac fibrosis in the remote LV tissue and expression of fibrosis markers collagen I and III, periostin and miR-21. Cardiomyocyte-specific ERß-OE improved survival associated with reduced maladaptive remodelling, improved cardiac function and less heart failure development after MI in both sexes. These effects seem to be related, at least in part, to a better maintenance of Ca(2+) cycling in both sexes and a lower induction of cardiac fibrosis in males after MI.


Assuntos
Receptor beta de Estrogênio/metabolismo , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Biomarcadores/metabolismo , Diástole/fisiologia , Receptor beta de Estrogênio/fisiologia , Feminino , Fibrose , Masculino , Camundongos Transgênicos , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Fatores Sexuais , Sístole/fisiologia , Ultrassonografia , Remodelação Ventricular/fisiologia
5.
Biochem Biophys Res Commun ; 449(3): 284-8, 2014 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-24857983

RESUMO

Binding of the utmost N-terminus of essential myosin light chains (ELC) to actin slows down myosin motor function. In this study, we investigated the binding constants of two different human cardiac ELC isoforms with actin. We employed circular dichroism (CD) and surface plasmon resonance (SPR) spectroscopy to determine structural properties and protein-protein interaction of recombinant human atrial and ventricular ELC (hALC-1 and hVLC-1, respectively) with α-actin as well as α-actin with alanin-mutated ELC binding site (α-actin(ala3)) as control. CD spectroscopy showed similar secondary structure of both hALC-1 and hVLC-1 with high degree of α-helicity. SPR spectroscopy revealed that the affinity of hALC-1 to α-actin (KD=575 nM) was significantly (p<0.01) lower compared with the affinity of hVLC-1 to α-actin (KD=186 nM). The reduced affinity of hALC-1 to α-actin was mainly due to a significantly (p<0.01) lower association rate (kon: 1,018 M(-1)s(-1)) compared with kon of the hVLC-1/α-actin complex interaction (2,908 M(-1)s(-1)). Hence, differential expression of ELC isoforms could modulate muscle contractile activity via distinct α-actin interactions.


Assuntos
Actinas/metabolismo , Miosinas Atriais/metabolismo , Cadeias Leves de Miosina/metabolismo , Miosinas Ventriculares/metabolismo , Actinas/química , Actinas/genética , Miosinas Atriais/química , Miosinas Atriais/genética , Dicroísmo Circular , Humanos , Contração Miocárdica , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/genética , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Miosinas Ventriculares/química , Miosinas Ventriculares/genética
6.
Biochem Biophys Res Commun ; 450(1): 464-9, 2014 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-24911555

RESUMO

The essential myosin light chain (ELC) is involved in modulation of force generation of myosin motors and cardiac contraction, while its mechanism of action remains elusive. We hypothesized that ELC could modulate myosin stiffness which subsequently determines its force production and cardiac contraction. Therefore, we generated heterologous transgenic mouse (TgM) strains with cardiomyocyte-specific expression of ELC with human ventricular ELC (hVLC-1; TgM(hVLC-1)) or E56G-mutated hVLC-1 (hVLC-1(E56G); TgM(E56G)). hVLC-1 or hVLC-1(E56G) expression in TgM was around 39% and 41%, respectively of total VLC-1. Laser trap and in vitro motility assays showed that stiffness and actin sliding velocity of myosin with hVLC-1 prepared from TgM(hVLC-1) (1.67 pN/nm and 2.3 µm/s, respectively) were significantly higher than myosin with hVLC-1(E56G) prepared from TgM(E56G) (1.25 pN/nm and 1.7 µm/s, respectively) or myosin with mouse VLC-1 (mVLC-1) prepared from C57/BL6 (1.41 pN/nm and 1.5 µm/s, respectively). Maximal left ventricular pressure development of isolated perfused hearts in vitro prepared from TgM(hVLC-1) (80.0 mmHg) were significantly higher than hearts from TgM(E56G) (66.2 mmHg) or C57/BL6 (59.3±3.9 mmHg). These findings show that ELCs decreased myosin stiffness, in vitro motility, and thereby cardiac functions in the order hVLC-1>hVLC-1(E56G)≈mVLC-1. They also suggest a molecular pathomechanism of hypertrophic cardiomyopathy caused by hVLC-1 mutations.


Assuntos
Coração/fisiologia , Contração Miocárdica/fisiologia , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Animais , Módulo de Elasticidade , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/fisiologia , Proteínas Motores Moleculares/ultraestrutura , Cadeias Leves de Miosina/ultraestrutura , Relação Estrutura-Atividade , Resistência à Tração/fisiologia
7.
J Biol Chem ; 286(11): 9079-96, 2011 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-21177871

RESUMO

A-kinase anchoring proteins (AKAPs) tether protein kinase A (PKA) and other signaling proteins to defined intracellular sites, thereby establishing compartmentalized cAMP signaling. AKAP-PKA interactions play key roles in various cellular processes, including the regulation of cardiac myocyte contractility. We discovered small molecules, 3,3'-diamino-4,4'-dihydroxydiphenylmethane (FMP-API-1) and its derivatives, which inhibit AKAP-PKA interactions in vitro and in cultured cardiac myocytes. The molecules bind to an allosteric site of regulatory subunits of PKA identifying a hitherto unrecognized region that controls AKAP-PKA interactions. FMP-API-1 also activates PKA. The net effect of FMP-API-1 is a selective interference with compartmentalized cAMP signaling. In cardiac myocytes, FMP-API-1 reveals a novel mechanism involved in terminating ß-adrenoreceptor-induced cAMP synthesis. In addition, FMP-API-1 leads to an increase in contractility of cultured rat cardiac myocytes and intact hearts. Thus, FMP-API-1 represents not only a novel means to study compartmentalized cAMP/PKA signaling but, due to its effects on cardiac myocytes and intact hearts, provides the basis for a new concept in the treatment of chronic heart failure.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Sistemas do Segundo Mensageiro/fisiologia , Proteínas de Ancoragem à Quinase A/genética , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Animais , Doença Crônica , AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Masculino , Contração Miocárdica/efeitos dos fármacos , Ratos , Ratos Endogâmicos WKY , Sistemas do Segundo Mensageiro/efeitos dos fármacos
8.
Biochem Biophys Res Commun ; 421(2): 184-9, 2012 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-22497893

RESUMO

Ahnak1 has been implicated in protein kinase A (PKA)-mediated control of cardiac L-type Ca(2+) channels (Cav1.2) through its interaction with the Cavß(2) regulatory channel subunit. Here we corroborate this functional linkage by immunocytochemistry on isolated cardiomyocytes showing co-localization of ahnak1 and Cavß(2) in the T-tubule system. In previous studies Cavß(2) attachment sites which impacted the channel's PKA regulation have been located to ahnak1's proximal C-terminus (ahnak1(4889-5535), ahnak1(5462-5535)). In this study, we mapped the ahnak1-interacting regions in Cavß(2) and investigated whether Cavß(2) phosphorylation affects its binding behavior. In vitro binding assays with Cavß(2) truncation mutants and ahnak1(4889-5535) revealed that the core region of Cavß(2) consisting of Src-homology 3 (SH3), HOOK, and guanylate kinase (GK) domains was important for ahnak1 interaction while the C- and N-terminal regions were dispensable. Furthermore, Ser-296 in the GK domain of Cavß(2) was identified as novel PKA phosphorylation site by mass spectrometry. Surface plasmon resonance (SPR) binding analysis showed that Ser-296 phosphorylation did not affect the high affinity interaction (K(D)≈35 nM) between Cavß(2) and the α(1C) I-II linker, but affected ahnak1 interaction in a complex manner. SPR experiments with ahnak1(5462-5535) revealed that PKA phosphorylation of Cavß(2) significantly increased the binding affinity and, in parallel, it reduced the binding capacity. Intriguingly, the phosphorylation mimic substitution Glu-296 fully reproduced both effects, increased the affinity by ≈2.4-fold and reduced the capacity by ≈60%. Our results are indicative for the release of a population of low affinity interaction sites following Cavß(2) phosphorylation on Ser-296. We propose that this phosphorylation event is one mechanism underlying ahnak1's modulator function on Cav1.2 channel activity.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteínas de Membrana/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas de Neoplasias/metabolismo , Serina/metabolismo , Animais , Canais de Cálcio Tipo L/genética , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Camundongos , Camundongos Endogâmicos , Fosforilação , Mapas de Interação de Proteínas , Estrutura Terciária de Proteína , Serina/genética
9.
Biochem Biophys Res Commun ; 405(3): 473-9, 2011 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-21256114

RESUMO

Myomesin plays an important structural and functional role in the M-band of striated muscles. The C-terminal domain 13 of myomesin dimerises and forms antiparallel strands which cross-link neighboring Myosin filaments and titin in the M-line of the sarcomeres. These interactions stabilise the contractile apparatus during striated muscle contraction. Since myomesin is an important component of the M-band we screened the myomesin gene for genetic variants in patients with hypertrophic cardiomyopathy (HCM). We identified the missense mutation V1490I in domain 12 of myomesin in a family with inherited HCM. Analytical ultracentrifugation experiments, circular dichroism spectra, and surface plasmon resonance spectroscopy of myomesin fragments were carried out to investigate the effects of the mutation V1490I on structure and function of myomesin domains 11-13 and 12-13. Both the wild type and mutated myomesin domains My11-13 revealed similar secondary structures and formed stable dimers. Mutated myomesin domains My11-13 and My12-13 dimers revealed a reduced thermal stability and a significantly decreased dimerisation affinity, showing disturbed functional properties of V1490I mutated myomesin. However, monomeric myomesin domains My11-12, i.e. without dimerisation domain 13 showed no difference in thermal stability between wild type and V1490I mutated myomesin. In conclusion, the V1490I mutation associated with HCM lead to myomesin proteins with abnormal functional properties which affect dimerisation properties of myomesin domain 13. These effects may contribute to the pathogenesis of HCM.


Assuntos
Cardiomiopatia Hipertrófica/genética , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Adulto , Conectina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Musculares/química , Mutação de Sentido Incorreto , Linhagem , Conformação Proteica , Multimerização Proteica , Ressonância de Plasmônio de Superfície
10.
J Muscle Res Cell Motil ; 32(4-5): 243-8, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21922228

RESUMO

Spinophilin (SPN) is a ubiquitously expressed scaffolding protein that interacts through several binding modules with a variety of target proteins. Thus, SPN bundles F-actin, targets protein phosphatase 1 to the ryanodine receptor, and targets regulators of G-protein signaling to G-protein coupled receptors in cardiomyocytes. In this work we studied the role of SPN on cardiomyocyte morphology, function, and ß-adrenergic responsiveness using a homozygous SPN knock-out mouse model (SPN-/-). We show that spinophilin deficiency significantly (1) reduced cardiomyocyte length, (2) increases both Ca(2+) amplitude and maximal rate of Ca(2+) rise during systole, and (3) decreased shortening amplitude and maximal rate of shortening, while (4) ß-adrenergic stimulation remained intact. Our data suggest that spinophilin is an upstream regulator required for normal growth and excitation-contraction coupling, but is dispensable for ß-adrenergic stimulation of adult cardiomyocytes.


Assuntos
Cálcio/metabolismo , Proteínas dos Microfilamentos/metabolismo , Miócitos Cardíacos , Proteínas do Tecido Nervoso/metabolismo , Norepinefrina/metabolismo , Receptores Adrenérgicos beta/metabolismo , Animais , Tamanho Celular , Proteínas de Ligação ao GTP/metabolismo , Homeostase , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Modelos Animais , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Transdução de Sinais/fisiologia , Sístole/fisiologia
11.
J Muscle Res Cell Motil ; 32(4-5): 271-80, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22057634

RESUMO

Ahnak1 is a giant, ubiquitously expressed, plasma membrane support protein whose function in skeletal muscle is largely unknown. Therefore, we investigated whether ahnak would be influenced by alterations of the sarcolemma exemplified by dysferlin mutations known to render the sarcolemma vulnerable or by mutations in calpain3, a protease known to cleave ahnak. Human muscle biopsy specimens obtained from patients with limb girdle muscular dystrophy (LGMD) caused by mutations in dysferlin (LGMD2B) and calpain3 (LGMD2A) were investigated for ahnak expression and localization. We found that ahnak1 has lost its sarcolemmal localization in LGMD2B but not in LGMD2A. Instead ahnak1 appeared in muscle connective tissue surrounding the extracellular site of the muscle fiber in both muscular dystrophies. The entire giant ahnak1 molecule was present outside the muscle fiber and did only partially colocalize with CD45-positive immune cell infiltration and the extracelluar matrix proteins fibronectin and collagenVI. Further, vesicles shedded in response to Ca(2+) by primary human myotubes were purified and their protein content was analysed. Ahnak1 was prominently present in these vesicles. Electron microscopy revealed a homogenous population of vesicles with a diameter of about 150 nm. This is the first study demonstrating vesicle release from human myotubes that may be one mechanism underlying abnormally localized ahnak1. Taken together, our results define ahnak1 in muscle connective tissue as a novel feature of two genetically distinct muscular dystrophies that might contribute to disease pathology.


Assuntos
Tecido Conjuntivo/ultraestrutura , Proteínas de Membrana/metabolismo , Fibras Musculares Esqueléticas/ultraestrutura , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Proteínas de Neoplasias/metabolismo , Sarcolema/ultraestrutura , Vesículas Transportadoras/ultraestrutura , Calpaína/genética , Calpaína/metabolismo , Estudos de Casos e Controles , Disferlina , Homozigoto , Humanos , Imuno-Histoquímica , Proteínas de Membrana/genética , Microscopia Eletrônica , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Distrofia Muscular do Cíngulo dos Membros/genética , Mutação , Vesículas Transportadoras/metabolismo
12.
J Muscle Res Cell Motil ; 32(4-5): 281-90, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22038483

RESUMO

Ahnak1 has been implicated in the beta-adrenergic regulation of the cardiac L-type Ca(2+) channel current (I (CaL)) by its binding to the regulatory Cavß(2) subunit. In this study, we addressed the question whether ahnak1/Cavß(2) interactions are essential or redundant for beta-adrenergic stimulation of I (CaL). Three naturally occurring ahnak1 variants (V5075 M, G5242R, and T5796 M) identified by genetic screening of cardiomyopathy patients did essentially not influence the in vitro Cavß(2) interaction as assessed by recombinant proteins. But, we observed a robust increase in Cavß(2) binding by mutating Ala at position 4984 to Pro which creates a PxxP consensus motif in the ahnak1 protein fragment. Surface plasmon resonance measurements revealed that this mutation introduced an additional Cavß(2) binding site. The functionality of A4984P was supported by the specific action of the Pro-containing ahnak1-derived peptide (P4984) in beta-adrenergic regulation of I (CaL). Patch clamp recordings on cardiomyocytes showed that intracellular perfusion of P4984 markedly reduced I (CaL) response to the beta-adrenergic agonist, isoprenaline, while the Ala-containing counterpart failed to affect I (CaL). Interestingly, I (CaL) of ahnak1-deficient cardiomyocytes was not affected by peptide application. Moreover, I (CaL) of ahnak1-deficient cardiomyocytes showed intact beta-adrenergic responsiveness. Similarly isolated ahnak1-deficient mouse hearts responded normally to adrenergic challenge. Our results indicate that ahnak1 is not essential for beta-adrenergic up-regulation of I (CaL) and cardiac contractility in mice. But, tuning ahnak1/Cavß(2) interaction provides a tool for modulating the beta-adrenergic response of I (CaL).


Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteínas de Membrana/metabolismo , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Proteínas de Neoplasias/metabolismo , Agonistas Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Motivos de Aminoácidos , Animais , Sítios de Ligação/fisiologia , Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Sinalização do Cálcio/fisiologia , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Hipertrófica/metabolismo , Estudos de Casos e Controles , Humanos , Isoproterenol/metabolismo , Isoproterenol/farmacologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/efeitos dos fármacos , Proteínas de Neoplasias/genética , Técnicas de Patch-Clamp , Polimorfismo de Nucleotídeo Único , Receptores Adrenérgicos beta/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Regulação para Cima/efeitos dos fármacos
13.
Circ Res ; 105(4): 326-34, 2009 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-19608978

RESUMO

RATIONALE: Adipocyte fatty acid-binding protein (FABP4) is a member of the intracellular lipid-binding protein family and is predominantly expressed in adipose tissue. Emerging evidence suggests that FABP4 plays a role in some aspects of the metabolic syndrome including the development of type 2 diabetes and atherosclerosis. We have recently reported that secretory products from human adipocytes directly and acutely depressed cardiac contractile function. OBJECTIVE: The purpose of this study was to identify this adipocyte-derived cardiodepressant factor. METHODS AND RESULTS: Through mass spectrometry and immunoblotting, we have identified this cardiodepressant factor as FABP4. FABP4 represents 1.8% to 8.1% of total protein secreted by adipocytes in extracellular medium. FABP4 acutely depressed shortening amplitude as well as intracellular systolic peak Ca(2+) in a dose-dependent manner in isolated rat cardiomyocytes. Heart-specific FABP isoform (FABP3) revealed a similar cardiodepressant effect. The N-terminal amino acids 1 to 20 of FABP4 could be identified as the most effective cardiodepressive domain. We could exclude any effect of FABP4 on action potential duration and L-type Ca(2+) current, suggesting a reduced excitation-contraction gain caused by FABP4 as the main inhibitory mechanism. CONCLUSION: We conclude that the release of FABP4 from adipocytes may be involved in the development of cardiac contractile dysfunction of obese subjects.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Proteínas de Ligação a Ácido Graxo/farmacologia , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Adipócitos/metabolismo , Adulto , Idoso , Animais , Aterosclerose/metabolismo , Cálcio/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Relação Dose-Resposta a Droga , Proteínas de Ligação a Ácido Graxo/isolamento & purificação , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Humanos , Masculino , Síndrome Metabólica/metabolismo , Pessoa de Meia-Idade , Obesidade/metabolismo , Ratos , Ratos Wistar
14.
J Cachexia Sarcopenia Muscle ; 12(5): 1249-1265, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34212535

RESUMO

BACKGROUND: Aging is associated with a progressive reduction in cellular function leading to poor health and loss of physical performance. Mitochondrial dysfunction is one of the hallmarks of aging; hence, interventions targeting mitochondrial dysfunction have the potential to provide preventive and therapeutic benefits to elderly individuals. Meta-analyses of age-related gene expression profiles showed that the expression of Ahnak1, a protein regulating several signal-transduction pathways including metabolic homeostasis, is increased with age, which is associated with low VO2MAX and poor muscle fitness. However, the role of Ahnak1 in the aging process remained unknown. Here, we investigated the age-related role of Ahnak1 in murine exercise capacity, mitochondrial function, and contractile function of cardiac and skeletal muscles. METHODS: We employed 15- to 16-month-old female and male Ahnak1-knockout (Ahnak1-KO) and wild-type (WT) mice and performed morphometric, biochemical, and bioenergetics assays to evaluate the effects of Ahnak1 on exercise capacity and mitochondrial morphology and function in cardiomyocytes and tibialis anterior (TA) muscle. A human left ventricular (LV) cardiomyocyte cell line (AC16) was used to investigate the direct role of Ahnak1 in cardiomyocytes. RESULTS: We found that the level of Ahnak1 protein is significantly up-regulated with age in the murine LV (1.9-fold) and TA (1.8-fold) tissues. The suppression of Ahnak1 was associated with improved exercise tolerance, as all aged adult Ahnak1-KO mice (100%) successfully completed the running programme, whereas approximately 31% male and 8% female WT mice could maintain the required running speed and distance. Transmission electron microscopic studies showed that LV and TA tissue specimens of aged adult Ahnak1-KO of both sexes have significantly more enlarged/elongated mitochondria and less small mitochondria compared with WT littermates (P < 0.01 and P < 0.001, respectively) at basal level. Further, we observed a shift in mitochondrial fission/fusion balance towards fusion in cardiomyocytes and TA muscle from aged adult Ahnak1-KO mice. The maximal and reserve respiratory capacities were significantly higher in cardiomyocytes from aged adult Ahnak1-KO mice compared with the WT counterparts (P < 0.05 and P < 0.01, respectively). Cardiomyocyte contractility and fatigue resistance of TA muscles were significantly increased in Ahnak1-KO mice of both sexes, compared with the WT groups. In vitro studies using AC16 cells have confirmed that the alteration of mitochondrial function is indeed a direct effect of Ahnak1. Finally, we presented Ahnak1 as a novel cardiac mitochondrial membrane-associated protein. CONCLUSIONS: Our data suggest that Ahnak1 is involved in age-related cardiac and skeletal muscle dysfunction and could therefore serve as a promising therapeutical target.


Assuntos
Mitocôndrias , Músculo Esquelético , Animais , Feminino , Masculino , Camundongos , Camundongos Knockout , Dinâmica Mitocondrial , Contração Muscular , Músculo Esquelético/metabolismo
15.
Pflugers Arch ; 460(4): 719-30, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20607281

RESUMO

Ahnak1, a giant 700 kDa protein, has been implicated in Ca(2+) signalling in various cells. Previous work suggested that the interaction between ahnak1 and Cavbeta(2) subunit plays a role in L-type Ca(2+) current (I (CaL)) regulation. Here, we performed structure-function studies with the most C-terminal domain of ahnak1 (188 amino acids) containing a PxxP consensus motif (designated as 188-PSTP) using ventricular cardiomyocytes isolated from rats, wild-type (WT) mice and ahnak1-deficient mice. In vitro binding studies revealed that 188-PSTP conferred high-affinity binding to Cavbeta(2) (K (d) approximately 60 nM). Replacement of proline residues by alanines (188-ASTA) decreased Cavbeta(2) affinity about 20-fold. Both 188-PSTP and 188-ASTA were functional in ahnak1-expressing rat and mouse cardiomyocytes during whole-cell patch clamp. Upon intracellular application, they increased the net Ca(2+) influx by enhancing I (CaL) density and/or increasing I (CaL) inactivation time course without altering voltage dependency. Specifically, 188-ASTA, which failed to affect I (CaL) density, markedly slowed I (CaL) inactivation resulting in a 50-70% increase in transported Ca(2+) during a 0 mV depolarising pulse. Both ahnak1 fragments also slowed current inactivation with Ba(2+) as charge carrier. By contrast, neither 188-PSTP nor 188-ASTA affected any I (CaL) characteristics in ahnak1-deficient mouse cardiomyocytes. Our results indicate that the presence of endogenous ahnak1 is required for tuning the voltage-dependent component of I (CaL) inactivation by ahnak1 fragments. We suggest that ahnak1 modulates the accessibility of molecular determinants in Cavbeta(2) and/or scaffolds selectively different beta-subunit isoforms in the heart.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteínas de Membrana/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas de Neoplasias/metabolismo , Motivos de Aminoácidos , Animais , Western Blotting , Sinalização do Cálcio/fisiologia , Masculino , Proteínas de Membrana/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas de Neoplasias/química , Técnicas de Patch-Clamp , Ratos , Ratos Wistar
16.
Biochem Biophys Res Commun ; 401(1): 143-8, 2010 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-20833135

RESUMO

The AHNAK scaffold PDZ-protein family is implicated in various cellular processes including membrane repair; however, AHNAK function and subcellular localization in skeletal muscle are unclear. We used specific AHNAK1 and AHNAK2 antibodies to analyzed the detailed localization of both proteins in mouse skeletal muscle. Co-localization of AHNAK1 and AHNAK2 with vinculin clearly demonstrates that both proteins are components of the costameric network. In contrast, no AHNAK expression was detected in the T-tubule system. A laser wounding assay with AHNAK1-deficient fibers suggests that AHNAK1 is not involved in membrane repair. Using atomic force microscopy (AFM), we observed a significantly higher transverse stiffness of AHNAK1⁻/⁻ fibers. These findings suggest novel functions of AHNAK proteins in skeletal muscle.


Assuntos
Módulo de Elasticidade , Proteínas de Membrana/metabolismo , Fibras Musculares Esqueléticas/química , Proteínas de Neoplasias/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Animais , Proteínas do Citoesqueleto , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos , Microscopia de Força Atômica , Fibras Musculares Esqueléticas/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética
17.
Biochem Biophys Res Commun ; 396(4): 939-43, 2010 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-20460111

RESUMO

The denuded IQ2 domain, i.e. myosin heavy chain not associated with regulatory light chains, exerts an inhibitory effect on myosin ATPase activity. In this study, we elaborated a structural explanation for this auto-inhibitory effect of IQ2 on myosin function. We employed analytical ultracentrifugation, circular dichroism, and surface plasmon resonance spectroscopy to investigate structural and functional properties of a myosin heavy chain (MYH) head-rod fragment aa664-915. MYH(664-915) was monomeric, adopted a closed shape, and bound essential myosin light chains (HIS-MLC-1) with low affinity to IQ1. Deletion of IQ2, however opened MYH(664-915). Four amino acids present in IQ2 could be identified to be responsible for this auto-inhibitory structural effect: alanine mutagenesis of I814, Q815, R819, and W827 stretched MYH(664-915) and increased 30-fold the binding affinity of HIS-MLC-1 to IQ1. In this study we show, that denuded IQ2 favours a closed conformation of myosin with a low HIS-MLC-1 binding affinity. The collapsed structure of myosin with denuded IQ2 could explain the auto-inhibitory effects of IQ2 on enzymatic activity of myosin.


Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Cadeias Pesadas de Miosina/química , Miosina Tipo II/antagonistas & inibidores , Animais , Dicroísmo Circular , Cadeias Pesadas de Miosina/genética , Cadeias Leves de Miosina/química , Estrutura Terciária de Proteína/genética , Ratos , Deleção de Sequência , Ressonância de Plasmônio de Superfície , Ultracentrifugação
18.
J Muscle Res Cell Motil ; 29(6-8): 181-4, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19247583

RESUMO

Obesity is a major risk factor for metabolic syndrome and cardiovascular disorders. Obesity related heart disease is the most serious complication of human obesity. Despite several investigations the pathophysiological mechanisms involved remain unclear. Latest studies have emphasized the importance of adipose tissue as a highly endocrine organ which releases a wide variety of biological active substances. In this context we have recently showed that adipose tissue exerts highly potent cardiodepressant activity with an acute effect directly on cardiomyocytes contraction, thus explaining the tight association between obesity and heart failure. Further experiments led to the assumption that the activity is a protein, but some well-known adipocyte-derived proteins could be excluded to be responsible for the effect on cardiomyocytes. In the present study we investigated the production/secretion of this adipocyte-derived negative inotropic activity in more detail.


Assuntos
Adipócitos/fisiologia , Tecido Adiposo Branco/fisiologia , Contração Miocárdica/fisiologia , Obesidade/metabolismo , Obesidade/fisiopatologia , Adipócitos/metabolismo , Tecido Adiposo Branco/metabolismo , Adulto , Idoso , Animais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Miócitos Cardíacos/fisiologia , Ratos , Ratos Wistar , Adulto Jovem
19.
J Mol Med (Berl) ; 85(12): 1405-12, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17717642

RESUMO

In contrast to immortal cell lines, primary cells are hardly susceptible to intracellular delivery methods such as transfection. In this study, we evaluated the direct delivery of several cell-permeable peptides under noninvasive conditions into living primary adult rat cardiomyocytes. We specifically monitored the functional effects of a cell-permeable peptide containing the 15 amino acid N-terminal peptide from human ventricular light chain-1 (VLC-1) on contraction and intracellular Ca2+ signals after electrical stimulation in primary adult cardiomyocytes. The transducible VLC-1 variant was taken up by cardiomyocytes within 5 min with more than 95% efficiency and localized to sarcomeric structures. Analysis of the functional effects of the cell-permeable VLC-1 revealed an enhancement of the intrinsic contractility of cardiomyocytes without affecting the intracellular Ca2+. Therefore, peptide transduction mediated by cell-penetrating peptides represents not only a unique strategy to enhance heart muscle function with no secondary effect on intracellular Ca2+ but also an invaluable tool for the modulation and manipulation of protein interactions in general and in primary cells.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cardiotônicos/farmacologia , Permeabilidade da Membrana Celular , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Cadeias Leves de Miosina/farmacologia , Fragmentos de Peptídeos/farmacologia , Miosinas Ventriculares/farmacologia , Animais , Cardiotônicos/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Estimulação Elétrica , Humanos , Microscopia Confocal , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/metabolismo , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Endogâmicos WKY , Sarcômeros/efeitos dos fármacos , Sarcômeros/metabolismo , Miosinas Ventriculares/metabolismo
20.
FASEB J ; 20(10): 1653-9, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16873888

RESUMO

The causal relationship between obesity and heart failure is broadly acknowledged; however, the pathophysiological mechanisms involved remain unclear. In this study we investigated whether human adipocytes secrete cardioactive substances that may affect cardiomyocyte contractility. We cultivated adipocytes obtained from human white adipose tissue and incubated isolated rat adult cardiomyocytes with adipocyte-conditioned or control medium. This is the first report to demonstrate that human adipocytes exhibit cardiodepressant activity with a direct and acute effect on cardiomyocyte contraction. This adipocyte-derived negative inotropic activity directly depresses shortening amplitude as well as intracellular systolic peak Ca2+ in cardiomyocytes within a few minutes. The adipocyte-derived cardiodepressant activity was dose-dependent and was completely blunted by heating or by trypsin digestion. Filtration of adipocyte-conditioned medium based on molecular mass characterized the cardiodepressant activity at between 10 and 30 kDa. In summary, adipose tissue exerts highly potent activity with an acute depressant effect directly on cardiomyocytes, which may well contribute to increased heart failure risk in overweight patients.


Assuntos
Adipócitos/fisiologia , Contração Miocárdica , Miócitos Cardíacos/fisiologia , Adipócitos/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados/análise , Insuficiência Cardíaca/etiologia , Humanos , Peso Molecular , Obesidade/complicações , Comunicação Parácrina , Ratos
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