RESUMO
Cooperative myosin binding to the thin filament is critical to regulation of cardiac and skeletal muscle contraction. This report delineates and fits to experimental data a new model of this process, in which specific tropomyosin-actin interactions are important, the tropomyosin-tropomyosin polymer is continuous rather than disjointed, and tropomyosin affects myosin-actin binding by shifting among three positions as in recent structural studies. A myosin- and tropomyosin-induced conformational change in actin is proposed, rationalizing the approximately 10,000-fold strengthening effect of myosin on tropomyosin-actin binding. Also, myosin S1 binding to regulated filaments containing mutant tropomyosins with internal deletions exhibited exaggerated cooperativity, implying an allosteric effect of tropomyosin on actin and allowing the effect's measurement. Comparisons among the mutants suggest the change in actin is promoted much more strongly by the middle of tropomyosin than by its ends. Regardless of calcium binding to troponin, this change in actin facilitates the shift in tropomyosin position to the actin inner domain, which is required for tight myosin-actin association. It also increases myosin-actin affinity 7-fold compared with the absence of troponin-tropomyosin. Finally, initiation of a shift in tropomyosin position is 100-fold more difficult than is its extension from one actin to the next, producing the myosin binding cooperativity that underlies cooperative activation of muscle contraction.
Assuntos
Actinas/química , Actinas/metabolismo , Miosinas/química , Miosinas/metabolismo , Tropomiosina/metabolismo , Animais , Bovinos , Cinética , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Fibras Musculares de Contração Rápida/fisiologia , Músculo Esquelético/fisiologia , Subfragmentos de Miosina/química , Subfragmentos de Miosina/metabolismo , Conformação Proteica , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Tropomiosina/químicaRESUMO
Striated muscle contraction is regulated by Ca2+ binding to troponin, which has a globular domain and an elongated tail attributable to the NH2-terminal portion of the bovine cardiac troponin T (TnT) subunit. Truncation of the bovine cardiac troponin tail was investigated using recombinant TnT fragments and subunits TnI and TnC. Progressive truncation of the troponin tail caused progressively weaker binding of troponin-tropomyosin to actin and of troponin to actin-tropomyosin. A sharp drop-off in affinity occurred with NH2-terminal deletion of 119 rather than 94 residues. Deletion of 94 residues had no effect on Ca2+-activation of the myosin subfragment 1-thin filament MgATPase rate and did not eliminate cooperative effects of Ca2+ binding. Troponin tail peptide TnT1-153 strongly promoted tropomyosin binding to actin in the absence of TnI or TnC. The results show that the anchoring function of the troponin tail involves interactions with actin as well as with tropomyosin and has comparable importance in the presence or absence of Ca2+. Residues 95-153 are particularly important for anchoring, and residues 95-119 are crucial for function or local folding. Because striated muscle regulation involves switching among the conformational states of the thin filament, regulatory significance for the troponin tail may arise from its prominent contribution to the protein-protein interactions within these conformations.
Assuntos
Fibras Musculares Esqueléticas/metabolismo , Mutação , Miocárdio/metabolismo , Troponina T/metabolismo , Actinas/metabolismo , Animais , Bovinos , DNA Complementar , Metabolismo Energético , Ligação Proteica , Deleção de Sequência , Tropomiosina/metabolismo , Troponina T/genéticaRESUMO
The myosin subfragment 1 (S1) MgATPase rate was measured using thin filaments with known extents of Ca2+ binding controlled by varying the ratio of native cardiac troponin versus an inhibitory troponin with a mutation in the sole regulatory Ca2+ binding site of troponin C. Fractional MgATPase activation was less than the fraction of troponins that bound Ca2+, implying a cooperative effect of bound Ca2+ on cross-bridge cycling. Addition of phalloidin did not alter cooperative effects between bound Ca2+ molecules in the presence or absence of myosin S1. When the myosin S1 concentration was raised sufficiently to introduce cooperative myosin-myosin effects, lower Ca2+ concentrations were needed to activate the MgATPase rate. MgATPase activation remained less than Ca2+ binding, implying a true, not just an apparent, increase in Ca2+ affinity. MgATPase activation by Ca2+ was more cooperative than could be explained by cooperativeness of overall Ca2+ binding, the discrepancy between Ca2+ binding and MgATPase activation, or interactions between myosins. The results suggest the thin filament-myosin S1 MgATPase cycle requires calcium binding to adjacent troponin molecules and that this binding is cooperatively promoted by a single cycling cross-bridge. This mechanism is a potential explanation for Ca2+-mediated regulation of cross-bridge kinetics in muscle fibers.
Assuntos
Citoesqueleto de Actina/metabolismo , ATPase de Ca(2+) e Mg(2+)/metabolismo , Cálcio/metabolismo , Modelos Biológicos , Miocárdio/metabolismo , Subfragmentos de Miosina/metabolismo , Troponina/metabolismo , Animais , Bovinos , Ligação ProteicaRESUMO
Cardiac thin filaments contain many troponin C (TnC) molecules, each with one regulatory Ca2+ binding site. A statistical mechanical model for the effects of these sites is presented and investigated. The ternary troponin complex was reconstituted with either TnC or the TnC mutant CBMII, in which the regulatory site in cardiac TnC (site II) is inactivated. Regardless of whether Ca2+ was present, CBMII-troponin was inhibitory in a thin filament-myosin subfragment 1 MgATPase assay. The competitive binding of [3H]troponin and [14C]CBMII-troponin to actin.tropomyosin was measured. In the presence of Mg2+ and low free Ca2+ they had equal affinities for the thin filament. When Ca274+ was added, however, troponin's affinity for the thin filament was 2.2-fold larger for the mutant than for the wild type troponin. This quantitatively describes the effect of regulatory site Ca2+ on troponin's affinity for actin.tropomyosin; the decrease in troponin-thin filament binding energy is small. Application of the theoretical model to the competitive binding data indicated that troponin molecules bind to interdependent rather than independent sites on the thin filament. Ca2+ binding to the regulatory site of TnC has a long-range rather than a merely local effect. However, these indirect TnC-TnC interactions are weak, indicating that the cooperativity of muscle activation by Ca2+ requires other sources of cooperativity.
Assuntos
Cálcio/metabolismo , Miocárdio/metabolismo , Troponina/metabolismo , Actinas/metabolismo , Animais , Sítios de Ligação/genética , Ligação Competitiva , Fenômenos Biofísicos , Biofísica , Bovinos , Técnicas In Vitro , Substâncias Macromoleculares , Camundongos , Miocárdio/química , Mutação Puntual , Ligação Proteica , Tropomiosina/metabolismo , Troponina/química , Troponina/genética , Troponina CRESUMO
We tested the hypothesis that coronary angiogenesis in response to chronic thyroxine (T4) treatment is not limited by age. Male Fischer 344 rats aged either 8 (young adult) or 24 (senescent) mo were studied after receiving either L-thyroxine (0.2 mg/kg sc) or vehicle for 2 mo. Heart weight-to-body weight ratio, compared with age-matched controls, increased by 47 and 44% in 8- and 24-mo T4 groups, respectively. Maximal myocardial perfusion per unit mass, measured in diastole-arrested, maximally dilated, isolated hearts, was similar in T4 rats and their age group controls; however, flow tended to be lower in senescent than in young adult rats. Thus the cross-sectional area of the coronary vessels grew in proportion to the increase in cardiac mass. Morphometric analyses, based on image analysis, showed that capillary length density was slightly lower in the midmyocardium but not the epimyocardium of the 24-mo T4 group compared with their age group controls. However, volume density, surface density, and intercapillary distance were not influenced by T4 treatment and the presence of cardiac hypertrophy. We conclude that in this model of cardiac hypertrophy 1) coronary vessel growth parallels the increase in ventricular mass, 2) capillaries grow by proliferation and an increase in diameter, and 3) vascular growth is not notably compromised during senescence.
Assuntos
Envelhecimento/fisiologia , Capilares/fisiopatologia , Cardiomegalia/fisiopatologia , Vasos Coronários/fisiopatologia , Hemodinâmica , Microcirculação/fisiopatologia , Tiroxina/toxicidade , Animais , Pressão Sanguínea , Peso Corporal , Capilares/efeitos dos fármacos , Capilares/crescimento & desenvolvimento , Cardiomegalia/induzido quimicamente , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/crescimento & desenvolvimento , Diástole , Coração/anatomia & histologia , Coração/crescimento & desenvolvimento , Masculino , Microcirculação/efeitos dos fármacos , Microcirculação/crescimento & desenvolvimento , Tamanho do Órgão , Ratos , Ratos Endogâmicos F344 , SístoleRESUMO
A major focus in studies of muscle contraction has been the effect of Ca2+ on the interactions among the thin filament's five constituent polypeptides: actin, tropomyosin, troponin C (TnC), troponin T (TnT), and troponin I (TnI). We have investigated these interactions by analyzing thin filament assembly as a linear lattice binding problem with linkage relationships in the associations of tropomyosin, actin, and troponin. Binding of TnT, the binary TnT.TnI complex, or the ternary troponin complex (+/- Ca2+) to tropomyosin was measured spectrofluorimetrically after labeling cardiac tropomyosin with N-(1-pyrene)iodoacetamide. The affinity constants ranged between 0.2 and 0.6 microM-1 in the presence of 300 mM KCl. Also, the affinities of tropomyosin, tropomyosin-troponin, tropomyosin.TnT, and tropomyosin.TnT.TnI for an isolated site on F-actin were determined. The actin association constants were 0.0006 microM-1 for tropomyosin, 1 microM-1 for tropomyosin.TnT, 2 microM-1 for tropomyosin.TnT.TnI, 0.5 microM-1 for tropomyosin.troponin, and 0.5 microM-1 for tropomyosin-troponin.Ca2+. Linked equilibrium analysis permitted calculation of the affinities for actin.tropomyosin of TnT (400 microM-1), TnT.TnI (1600 microM-1), troponin (500 microM-1), and troponin.Ca2+ (300 microM-1). Therefore, both troponin and tropomyosin.troponin retain high actin-affinity even when Ca2+ is present or when TnI is removed, and even in the absence of cooperative contributions. The results are discussed in consideration of increasing evidence for a Ca(2+)-regulated azimuthal movement of tropomyosin on F-actin (Lehman, W., Craig, R., and Vibert, P. (1994) Nature 368, 65-67). It is proposed that tropomyosin movement may be due to switching between TnI-mediated and TnT-mediated binding of troponin-tropomyosin to distinct sites on F-actin.
Assuntos
Proteínas dos Microfilamentos/fisiologia , Contração Miocárdica , Actinas/metabolismo , Animais , Bovinos , Proteínas dos Microfilamentos/química , Ligação Proteica , Coelhos , Tropomiosina/metabolismo , Troponina/metabolismoRESUMO
Recent analyses of the assembly of thin filaments containing altered forms of troponin (or no troponin) suggested that the strongly cooperative nature of troponin-tropomyosin binding to actin might be primarily caused by indirect interactions involving the actin lattice, rather than by direct contacts between neighboring troponin-tropomyosin molecules. To test this hypothesis, thin filament assembly was examined using either cardiac tropomyosin digested with carboxypeptidase A (cbpTm) or a tropomyosin with defective function at both amino and carboxyl termini (unacetylated cbpTm). Compared to intact troponin-tropomyosin, both troponin-cbpTm and troponin-unacetylated cbpTm had much weaker binding to actin; however, cooperative interactions were only slightly reduced. These data support the implication that the primary source of the cooperativity involves troponin-tropomyosin-promoted conformational changes within the actin polymer. Surprisingly, the effects of tropomyosin amino- and carboxyl-terminal structural defects on troponin-tropomyosin binding to actin were not additive. In the presence of troponin, tropomyosin molecules with either defect had the same diminution in actin affinity as molecules with both defects. Finally, the Ca2+ sensitivity of troponin-tropomyosin binding to actin was increased by alteration of either end of tropomyosin.
Assuntos
Actinas/metabolismo , Tropomiosina/metabolismo , Troponina/metabolismo , Animais , Sítios de Ligação , Cálcio/metabolismo , Carboxipeptidases/farmacologia , Carboxipeptidases A , Músculos/metabolismo , Concentração Osmolar , Coelhos , RatosRESUMO
We examined the role of age in the initiation of thyroxine-induced (T4) cardiac hypertrophy. T4 (0.4 mg/kg sc) was administered to prepubescent (2 mo), young adult (6 mo), and senescent (24 mo) Fischer 344 rats for 4 days. While significant increases in left ventricular (LV) mass and RNA/LV were evident at 4 days in all T4-treated groups, the elevation in RNA/LV occurred earlier (2 days) in the senescent group. A 0.2-mg/kg dose of T4 elevated RNA values within 24 h in senescent, but not in prepubescent, rats. LV norepinephrine levels were measured to determine whether it plays a role in this model of cardiac hypertrophy. When synthesis of this catecholamine was blocked with alpha-methyl-p-tyrosine, tissue levels fell significantly in all groups, and the decrement was similar in T4-treated and control rats in the two younger groups. We conclude that: 1) the initiation of T4-induced cardiac hypertrophy is not compromised in senescent rats, 2) hearts of senescent rats respond earlier and to a lower dose of T4 than young rats, and 3) the cardiac hypertrophy that occurs in hyperthyroidism is not due to enhanced levels of available cardiac norepinephrine.
Assuntos
Envelhecimento/fisiologia , Hipertrofia Ventricular Esquerda/induzido quimicamente , Tiroxina/efeitos adversos , Animais , DNA/análise , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Coração/fisiologia , Hemodinâmica/fisiologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Miocárdio/química , Miocárdio/patologia , Norepinefrina/análise , Norepinefrina/fisiologia , Ratos , Ratos Endogâmicos F344RESUMO
Bacterially expressed alpha-tropomyosin lacks the amino-terminal acetylation present in muscle tropomyosin and binds poorly to actin (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). Using a linear lattice model, we determined the affinity (Ko) of unacetylated tropomyosin or troponin-unacetylated tropomyosin for an isolated site on the actin filament and the fold increase in affinity (y) when binding is to an adjacent site. The absence of tropomyosin acetylation decreased Ko 2 orders of magnitude in the absence of troponin. Tropomyosin acetylation also enhanced troponin-tropomyosin binding to actin, not by increasing cooperativity (y), but rather by increasing Ko. These results suggest that the amino-terminal region of tropomyosin is a crucial actin binding site. Troponin promoted unacetylated tropomyosin binding to actin, increasing Ko more than 1,000-fold. Troponin70-259, which lacks the troponin T peptide (1-69) spanning the overlap between adjacent tropomyosins, behaved similarly to intact troponin. Cooperative interactions between adjacent troponin-tropomyosin complexes remained strong despite the use of a nonpolymerizable tropomyosin and a troponin unable to bridge neighboring tropomyosins physically. The Ko for troponin70-259-unacetylated tropomyosin was 500-fold greater than for troponin159-259-unacetylated tropomyosin, indicating that troponin T residues 70-158 are critical for anchoring troponin-tropomyosin to F-actin. The mechanism of cooperative thin filament assembly is discussed.
Assuntos
Tropomiosina/metabolismo , Troponina/metabolismo , Acetilação , Actinas/isolamento & purificação , Actinas/metabolismo , Animais , Sítios de Ligação , Clonagem Molecular , DNA/genética , DNA/metabolismo , Cinética , Matemática , Músculos/metabolismo , Subfragmentos de Miosina/isolamento & purificação , Subfragmentos de Miosina/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Tropomiosina/genética , Tropomiosina/isolamento & purificação , Troponina/isolamento & purificação , Troponina TRESUMO
The binding of tropomyosin to actin and troponin-tropomyosin to actin was analyzed according to a linear lattice model which quantifies two parameters: Ko, the affinity of the ligand for an isolated site on the actin filament, and gamma, the fold increase in affinity when binding is contiguous to an occupied site (cooperativity). Tropomyosin-actin binding is very cooperative (gamma = 90-137). Troponin strengthens tropomyosin-actin binding greatly but, surprisingly, does so solely by an 80-130-fold increase in Ko, while cooperativity actually decreases. Additionally, troponin complexes containing TnT subunits with deletions of either amino acids 1-69 (troponin70-259) or 1-158 (troponin159-259) were examined. Deletion of amino acids 1-69 had only small effects on Ko and y, despite this peptide's location spanning the joint between adjacent tropomyosins. Ca2+ reduced Ko by half for both troponin and troponin70-159 and had no detectable effect on cooperativity. Troponin159-259 had much weaker effects on tropomyosin-actin binding than did troponin70-259 and had no effect at all in the presence of Ca2+. This suggests the importance of Ca(2+)-insensitive interactions between tropomyosin and troponin T residues 70-159. Cooperativity was slightly lower for troponin159-259 than tropomyosin alone, suggesting that the globular head region of troponin affects tropomyosin-tropomyosin interactions along the thin filament.
Assuntos
Actinas/metabolismo , Músculos/metabolismo , Tropomiosina/metabolismo , Troponina/metabolismo , Actinas/isolamento & purificação , Animais , Cálcio/farmacologia , Eletroforese em Gel de Poliacrilamida , Cinética , Peso Molecular , Ligação Proteica , Coelhos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Tropomiosina/isolamento & purificação , Troponina/isolamento & purificaçãoRESUMO
We tested the hypothesis that late-onset hypertension in middle-aged (15 mo) and senescent (24 mo) rats would adversely affect the coronary microvasculature. Morphometric analyses were performed on coronary capillaries and arterioles from rats with one-kidney, figure-8 renal wrap hypertension of 3-mo duration. Compared with control rats, wall-to-lumen ratios of arterioles with lumen diameters less than 25 microns were higher in the two hypertensive groups by approximately 30%; larger arterioles did not show consistent intergroup differences. A comparison of the two control groups revealed that wall-to-lumen ratio of arterioles with lumen diameters less than 50 microns tended to be greater in the senescent rats. Capillary numerical density was markedly reduced in the hypertensive animals of both age groups and caused an increase in the mean Krough cylinder radius and in the mean capillary domain. The latter increased by 28-63%; the largest increment occurred in the endomyocardium of the senescent group. A trend toward increased heterogeneity of capillary spacing was also noted in the hypertensive rats. The observed microvascular alterations occurred in the absence of an absolute increase in left ventricular mass but in the presence of cardiocyte hypertrophy. Thus the decrements in capillary numerical density are not only due to inadequate growth but reflect an absolute reduction in the number of these vessels associated with cardiocyte loss. It is concluded that late-onset hypertension in middle-aged and senescent rats is characterized by left ventricular wall remodeling that includes microvascular alterations that would be expected to limit maximal myocardial flow and O2 supply to the cardiocyte.
Assuntos
Envelhecimento/fisiologia , Circulação Coronária , Hipertensão Renal/fisiopatologia , Animais , Arteríolas/patologia , Capilares/patologia , Coração/fisiopatologia , Ventrículos do Coração , Hipertensão Renal/patologia , Microcirculação , Miocárdio/patologia , RatosRESUMO
We have characterized ATP-dependent Ca2+ transport into highly purified plasma membrane fraction isolated from guinea pig ileum smooth muscle. The membrane fraction contained inside-out sealed vesicles and was enriched 30-40-fold in 5'-nucleotidase and phosphodiesterase I activity as compared to post nuclear supernatant. Plasma membrane vesicles showed high rate (76 nmol/mg/min) and high capacity for ATP dependent Ca2+ transport which was inhibited by addition of Ca2+ ionophore A23187. The inhibitors of mitochondrial Ca2+ transport, i.e., sodium azide, oligomycin and ruthenium red did not inhibit ATP-dependent Ca2+ uptake into plasma membrane vesicles. The energy dependent Ca2+ uptake into plasma membranes showed very high specificity for ATP as energy source and other nucleotide triphosphates were ineffective in supporting Ca2+ transport. Phosphate was significantly better as Ca2+ trapping anion to potentiate ATP-dependent Ca2+ uptake into plasma membrane fraction as compared to oxalate. Orthovanadate, an inhibitor of cell membrane (Ca2+-Mg2+)-ATPase activity, completely inhibited ATP-dependent Ca2+ transport and the Ki was approximately 0.6 microM. ATP-dependent Ca2+ transport and formation of alkali labile phosphorylated intermediate of (Ca2+-Mg2+)-ATPase increased with increasing concentrations of free Ca2+ in the incubation mixture and the Km value for Ca2+ was approximately 0.6-0.7 microM for both the reactions.
Assuntos
Cálcio/metabolismo , Íleo/metabolismo , Músculo Liso/metabolismo , Animais , ATPase de Ca(2+) e Mg(2+)/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/metabolismo , Cobaias , Cinética , Masculino , Ouabaína/farmacologia , Fosforilação , Vanadatos , Vanádio/farmacologiaRESUMO
Spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were used to investigate the adaptive biochemical changes in the myocardium in response to chronic afterload. Ouabain-inhibited Na+,K+-adenosine triphosphatase (ATPase) activity was decreased by 40% in myocardium of SHR compared with that from WKY, which may lead to increased intracellular Ca2+ through Na+-Ca2+ exchange. Similarly, alpha 1-adrenergic receptor density, estimated by [3H]prazosin binding, was decreased by 42% in myocardial membranes of SHR, while the affinity for the agonist and the antagonist was not altered. In contrast, the number of Ca2+ channels estimated by [3H]nitrendipine binding was increased by 45% in myocardial membranes of SHR, while the affinity was comparable between SHR and WKY. These differences between WKY and SHR in the membrane properties were not due to differential contamination of plasma membranes because the activities of other putative plasma membrane marker enzymes were comparable between WKY and SHR. There were no differences between WKY and SHR in the myosin ATPase activity estimated using myofibrils, actomyosin, and myosin. These results suggest that specific alterations have occurred in the plasma membrane properties of myocardium of SHR that result in altered intracellular Ca2+ metabolism. These alterations may have an important bearing on excitation-contraction coupling in myocardium of SHR.