RESUMO
A stacking sodium dodecyl sulfate polyacrylamide gel electrophoresis system has been used to resolve and quantify all the major myofibrillar protein components (actin, myosin, tropomyosin, and troponin C, T, and I). Quantification was achieved by densitometry of the fast green-stained gels calibrated with the use of purified proteins. The approximate molar ratios of these proteins in rabbit muscle are: actin : myosin: tropomyosin: troponin T: troponin I: troponin C = 7:1:1:1:1:1. On the basis of these results and available structural information one obtains an estimate of 254 myosin molecules per thick filament.
Assuntos
Miofibrilas , Tropomiosina , Actinas/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Músculo Esquelético/metabolismo , Miofibrilas/metabolismo , Miosinas/metabolismo , Coelhos , Tropomiosina/metabolismo , Troponina C/metabolismoRESUMO
Human slow skeletal troponin T (HSSTnT) shares a high degree of homology with cardiac TnT (CTnT). Although the presence of HSSTnT has not been confirmed in the heart at the protein level, detectable levels of HSSTnT mRNA have been found. Whether HSSTnT isoforms are expressed transiently remains unknown. Because transient re-expression of HSSTnT may be a potential mechanism of regulating function, we explored the effect of HSSTnT on the regulation of cardiac muscle. At least three HSSTnT isoforms have been found to exist in slow skeletal muscle: HSSTnT1 (+exons 5 and 12), HSSTnT2 (+exon 5, -exon 12), and HSSTnT3 (-exons 5 and 12). Another isoform, HSSTnT hypothetical (Hyp) (-exon 5, +exon 12), has only been found at the mRNA level. Compared with HCTnT3 (adult isoform), Tn complexes containing HSSTnT1, -2, and -3 did not alter the actomyosin ATPase activation and inhibition in the presence and absence of Ca(2+), respectively. HSSTnTHyp was not evaluated as it did not form a Tn complex under a variety of conditions. Porcine papillary skinned fibers displaced with HSSTnT1, -2, or -3 and reconstituted with human cardiac troponin I and troponin C (HCTnI·TnC) complex showed a decrease in the Ca(2+) sensitivity of force development and an increase in maximal recovered force (HSSTnT1 and -3) compared with HCTnT3. In contrast, HSSTnTHyp showed an increase in the Ca(2+) sensitivity of force development. This suggests that re- or overexpression of specific SSTnT isoforms might have therapeutic potential in the failing heart because they increase the maximal force of contraction. In addition, circular dichroism and proteolytic digestion experiments revealed structural differences between HSSTnT isoforms and HCTnT3 and that HSSTnT1 is more susceptible to calpain and trypsin proteolysis than the other HSSTnTs. Overall, HSSTnT isoforms despite being homologues of CTnT may display distinct functional properties in muscle regulation.
Assuntos
Contração Miocárdica , Miocárdio/citologia , Miócitos Cardíacos/fisiologia , Troponina T/fisiologia , Animais , Cálcio/fisiologia , Calpaína/química , Dicroísmo Circular , Humanos , Técnicas In Vitro , Miocárdio/enzimologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miosinas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Estrutura Secundária de Proteína , Proteólise , Sus scrofa , Troponina T/química , Troponina T/metabolismo , Tripsina/químicaRESUMO
Defined as clinically unexplained hypertrophy of the left ventricle, hypertrophic cardiomyopathy (HCM) is traditionally understood as a disease of the cardiac sarcomere. Mutations in TNNC1-encoded cardiac troponin C (cTnC) are a relatively rare cause of HCM. Here, we report clinical and functional characterization of a novel TNNC1 mutation, A31S, identified in a pediatric HCM proband with multiple episodes of ventricular fibrillation and aborted sudden cardiac death. Diagnosed at age 5, the proband is family history-negative for HCM or sudden cardiac death, suggesting a de novo mutation. TnC-extracted cardiac skinned fibers were reconstituted with the cTnC-A31S mutant, which increased Ca(2+) sensitivity with no effect on the maximal contractile force generation. Reconstituted actomyosin ATPase assays with 50% cTnC-A31S:50% cTnC-WT demonstrated Ca(2+) sensitivity that was intermediate between 100% cTnC-A31S and 100% cTnC-WT, whereas the mutant increased the activation of the actomyosin ATPase without affecting the inhibitory qualities of the ATPase. The secondary structure of the cTnC mutant was evaluated by circular dichroism, which did not indicate global changes in structure. Fluorescence studies demonstrated increased Ca(2+) affinity in isolated cTnC, the troponin complex, thin filament, and to a lesser degree, thin filament with myosin subfragment 1. These results suggest that this mutation has a direct effect on the Ca(2+) sensitivity of the myofilament, which may alter Ca(2+) handling and contribute to the arrhythmogenesis observed in the proband. In summary, we report a novel mutation in the TNNC1 gene that is associated with HCM pathogenesis and may predispose to the pathogenesis of a fatal arrhythmogenic subtype of HCM.
Assuntos
Cardiomiopatia Hipertrófica/genética , Predisposição Genética para Doença , Mutação , Miocárdio/metabolismo , Troponina C/genética , Troponina C/metabolismo , Fibrilação Ventricular/genética , Alelos , Sítios de Ligação , Cálcio/química , Cálcio/metabolismo , Cardiomiopatia Hipertrófica/fisiopatologia , Dicroísmo Circular , Clonagem Molecular , Estudos de Coortes , Humanos , Conformação Molecular , Miofibrilas/metabolismo , Miosinas/química , Fibrilação Ventricular/fisiopatologiaRESUMO
The R21C substitution in cardiac troponin I (cTnI) is the only identified mutation within its unique N-terminal extension that is associated with hypertrophic cardiomyopathy (HCM) in man. Particularly, this mutation is located in the consensus sequence for ß-adrenergic-activated protein kinase A (PKA)-mediated phosphorylation. The mechanisms by which this mutation leads to heart disease are still unclear. Therefore, we generated cTnI knock-in mouse models carrying an R21C mutation to evaluate the resultant functional consequences. Measuring the in vivo levels of incorporated mutant and WT cTnI, and their basal phosphorylation levels by top-down mass spectrometry demonstrated: 1) a dominant-negative effect such that, the R21C+/- hearts incorporated 24.9% of the mutant cTnI within the myofilament; and 2) the R21C mutation abolished the in vivo phosphorylation of Ser(23)/Ser(24) in the mutant cTnI. Adult heterozygous (R21C+/-) and homozygous (R21C+/+) mutant mice activated the fetal gene program and developed a remarkable degree of cardiac hypertrophy and fibrosis. Investigation of cardiac skinned fibers isolated from WT and heterozygous mice revealed that the WT cTnI was completely phosphorylated at Ser(23)/Ser(24) unless the mice were pre-treated with propranolol. After propranolol treatment (-PKA), the pCa-tension relationships of all three mice (i.e. WT, R21C+/-, and R21C+/+) were essentially the same. However, after treatment with propranolol and PKA, the R21C cTnI mutation reduced (R21C+/-) or abolished (R21C+/+) the well known decrease in the Ca(2+) sensitivity of tension that accompanies Ser(23)/Ser(24) cTnI phosphorylation. Altogether, the combined effects of the R21C mutation appear to contribute toward the development of HCM and suggest that another physiological role for the phosphorylation of Ser(23)/Ser(24) in cTnI is to prevent cardiac hypertrophy.
Assuntos
Substituição de Aminoácidos , Cardiomiopatia Hipertrófica Familiar/metabolismo , Mutação de Sentido Incorreto , Miocárdio/metabolismo , Miofibrilas/metabolismo , Troponina I/metabolismo , Animais , Antiarrítmicos/farmacologia , Cálcio/metabolismo , Cardiomiopatia Hipertrófica Familiar/genética , Cardiomiopatia Hipertrófica Familiar/patologia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fibrose Endomiocárdica/genética , Fibrose Endomiocárdica/metabolismo , Técnicas de Introdução de Genes , Humanos , Camundongos , Camundongos Mutantes , Miocárdio/patologia , Miofibrilas/genética , Miofibrilas/patologia , Fosforilação/genética , Propranolol/farmacologia , Troponina I/genéticaRESUMO
Mutations in TNNT2, encoding cardiac troponin T, commonly shows early onset, aggressive dilated cardiomyopathy (DCM). This observation may influence the decision of whether to undertake clinical genetic testing for TNNT2 in later onset DCM. Further, the trigger for late onset DCM remains enigmatic. A 70-year-old woman, previously healthy with a left ventricular ejection fraction of 50%-55% at age 69, presented with DCM of unknown cause and a 4-month history progressive heart failure requiring cardiac transplantation. Clinical genetic testing revealed a novel TNNT2 R139H mutation but no relevant variants in 18 other DCM genes. Her explanted heart showed partial fatty replacement in the right ventricle. Sequencing for five arrhythmogenic right ventricular dysplasia genes was negative. Functional studies in porcine cardiac skinned fibers reconstituted with the mutant R139H troponin T protein showed decreased Ca(2+) sensitivity at pH 7, characteristic of DCM. Because fatty infiltration may acidify the myocellular environment, maximal force development examined at pH 6.5 was diminished, suggesting a possible environmental trigger. We conclude that the TNNT2 R139H mutation was likely to be disease causing. Further, later age of onset may not be relevant to exclude genetic testing for TNNT2 mutations.
Assuntos
Cardiomiopatia Dilatada/genética , Mutação/genética , Miocárdio/patologia , Troponina T/genética , Idade de Início , Idoso , Substituição de Aminoácidos/genética , Animais , Cardiomiopatia Dilatada/patologia , Éxons/genética , Feminino , Heterozigoto , Humanos , Íntrons/genética , Masculino , Linhagem , Sus scrofaRESUMO
Cardiac diseases associated with mutations in troponin subunits include hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and restrictive cardiomyopathy (RCM). Altered calcium handling in these diseases is evidenced by changes in the Ca(2+) sensitivity of contraction. Mutations in the Ca(2+) sensor, troponin C (TnC), were generated to increase/decrease the Ca(2+) sensitivity of cardiac skinned fibers to create the characteristic effects of DCM, HCM, and RCM. We also used a reconstituted assay to determine the mutation effects on ATPase activation and inhibition. One mutant (A23Q) was found with HCM-like properties (increased Ca(2+) sensitivity of force and normal levels of ATPase inhibition). Three mutants (S37G, V44Q, and L48Q) were identified with RCM-like properties (a large increase in Ca(2+) sensitivity, partial loss of ATPase inhibition, and increased basal force). Two mutations were identified (E40A and I61Q) with DCM properties (decreased Ca(2+) sensitivity, maximal force recovery, and activation of the ATPase at high [Ca(2+)]). Steady-state fluorescence was utilized to assess Ca(2+) affinity in isolated cardiac (c)TnCs containing F27W and did not necessarily mirror the fiber Ca(2+) sensitivity. Circular dichroism of mutant cTnCs revealed a trend where increased alpha-helical content correlated with increased Ca(2+) sensitivity in skinned fibers and vice versa. The main findings from this study were as follows: 1) cTnC mutants demonstrated distinct functional phenotypes reminiscent of bona fide HCM, RCM, and DCM mutations; 2) a region in cTnC associated with increased Ca(2+) sensitivity in skinned fibers was identified; and 3) the F27W reporter mutation affected Ca(2+) sensitivity, maximal force, and ATPase activation of some mutants.
Assuntos
Cálcio/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Fenótipo , Troponina C/metabolismo , Actomiosina/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Cálcio/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Camundongos , Modelos Moleculares , Fibras Musculares Esqueléticas/metabolismo , Mutação , Contração Miocárdica/efeitos dos fármacos , Engenharia de Proteínas , Estrutura Secundária de Proteína , Coelhos , Troponina C/química , Troponina C/genéticaRESUMO
In this study we explore the mechanisms by which a double mutation (E59D/D75Y) in cardiac troponin C (CTnC) associated with dilated cardiomyopathy reduces the Ca(2+)-activated maximal tension of cardiac muscle. Studying the single mutants (i.e. E59D or D75Y) indicates that D75Y, but not E59D, causes a reduction in the calcium affinity of CTnC in troponin complex, regulated thin filaments (RTF), and the Ca(2+) sensitivity of contraction and ATPase in cardiac muscle preparations. However, both D75Y and E59D are required to reduce the actomyosin ATPase activity and maximal force in muscle fibers, indicating that E59D enhances the effects of D75Y. Part of the reduction in force/ATPase may be due to a defect in the interactions between CTnC and cardiac troponin T, which are known to be necessary for ATPase activation. An additional mechanism for the reduction in force/ATPase comes from measurements of the binding stoichiometry of myosin subfragment-1 (S-1) to the RTF. Using wild type RTFs, 4.8 mol S-1 was bound per mol filament (seven actins), whereas with E59D/D75Y RTFs, the number of binding sites was reduced by approximately 23% to 3.7. Altogether, these results suggest that the reduction in force and ATPase activation is possibly due to a thin filament conformation that promotes fewer accessible S-1-binding sites. In the absence of any family segregation data, the functional results presented here support the concept that this is likely a dilated cardiomyopathy-causing mutation.
Assuntos
Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Mutação , Troponina C/genética , Actinas/química , Adenosina Trifosfatases/química , Animais , Sítios de Ligação , Cálcio/química , Ativação Enzimática , Humanos , Contração Muscular , Miosinas/química , Ligação Proteica , Conformação Proteica , SuínosRESUMO
The human cardiac troponin I (hcTnI) mutation R145W has been associated with restrictive cardiomyopathy. In this study, simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145W transgenic mice (Tg-R145W) were explored. Tg-R145W fibers showed an approximately 13-16% increase in maximal Ca(2+)-activated force and ATPase activity compared to hcTnI wild-type transgenic mice. The force-generating cross-bridge turnover rate (g) and the energy cost (ATPase/force) were the same in all groups of fibers. Also, the Tg-R145W fibers showed a large increase in the Ca(2+) sensitivity of both force development and ATPase. In intact fibers, the mutation caused prolonged force and intracellular [Ca(2+)] transients and increased time to peak force. Analysis of force and Ca(2+) transients showed that there was a 40% increase in peak force in Tg-R145W muscles, which was likely due to the increased Ca(2+) transient duration. The above cited results suggest that: (1) there would be an increase in resistance to ventricular filling during diastole resulting from the prolonged force and Ca(2+) transients that would result in a decrease in ventricular filling (diastolic dysfunction); and (2) there would be a large (approximately 53%) increase in force during systole, which may help to partly compensate for diastolic dysfunction. These functional results help to explain the mechanisms by which these mutations give rise to a restrictive phenotype.
Assuntos
Substituição de Aminoácidos/genética , Cardiomiopatia Restritiva/genética , Mutação de Sentido Incorreto , Troponina I/genética , Trifosfato de Adenosina/metabolismo , Animais , Humanos , Camundongos , Camundongos Transgênicos , Contração Miocárdica , Miofibrilas/fisiologiaRESUMO
Recently four new hypertrophic cardiomyopathy mutations in cardiac troponin C (cTnC) (A8V, C84Y, E134D, and D145E) were reported, and their effects on the Ca(2+) sensitivity of force development were evaluated (Landstrom, A. P., Parvatiyar, M. S., Pinto, J. R., Marquardt, M. L., Bos, J. M., Tester, D. J., Ommen, S. R., Potter, J. D., and Ackerman, M. J. (2008) J. Mol. Cell. Cardiol. 45, 281-288). We performed actomyosin ATPase and spectroscopic solution studies to investigate the molecular properties of these mutations. Actomyosin ATPase activity was measured as a function of [Ca(2+)] utilizing reconstituted thin filaments (TFs) with 50% mutant and 50% wild type (WT) and 100% mutant cardiac troponin (cTn) complexes: A8V, C84Y, and D145E increased the Ca(2+) sensitivity with only A8V demonstrating lowered Ca(2+) sensitization at the 50% ratio when compared with 100%; E134D was the same as WT at both ratios. Of these four mutants, only D145E showed increased ATPase activation in the presence of Ca(2+). None of the mutants affected ATPase inhibition or the binding of cTn to the TF measured by co-sedimentation. Only D145E increased the Ca(2+) affinity of site II measured by 2-(4'-(2''-iodoacetamido)phenyl)aminonaphthalene-6-sulfonic acid fluorescence in isolated cTnC or the cTn complex. In the presence of the TF, only A8V was further sensitized to Ca(2+). Circular dichroism measurements in different metal-bound states of the isolated cTnCs showed changes in the secondary structure of A8V, C84Y, and D145E, whereas E134D was the same as WT. PyMol modeling of each cTnC mutant within the cTn complex revealed potential for local changes in the tertiary structure of A8V, C84Y, and D145E. Our results indicate that 1) three of the hypertrophic cardiomyopathy cTnC mutants increased the Ca(2+) sensitivity of the myofilament; 2) the effects of the mutations on the Ca(2+) affinity of isolated cTnC, cTn, and TF are not sufficient to explain the large Ca(2+) sensitivity changes seen in reconstituted and fiber assays; and 3) changes in the secondary structure of the cTnC mutants may contribute to modified protein-protein interactions along the sarcomere lattice disrupting the coupling between the cross-bridge and Ca(2+) binding to cTnC.
Assuntos
Cardiomiopatia Hipertrófica/genética , Mutação , Troponina C/genética , Actinas/química , Adenosina Trifosfatases/química , Sítios de Ligação , Cálcio/química , Cardiomiopatia Hipertrófica/patologia , Dicroísmo Circular , Clonagem Molecular , Humanos , Conformação Molecular , Miocárdio/patologia , Ligação Proteica , Estrutura Terciária de Proteína , Sensibilidade e Especificidade , Troponina C/fisiologiaRESUMO
In this study, we addressed the functional consequences of the human cardiac troponin I (hcTnI) hypertrophic cardiomyopathy R145G mutation in transgenic mice. Simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145G transgenic mice (Tg-R145G) versus hcTnI wild type transgenic mice (Tg-WT) showed a significant decrease in the maximal Ca(2+)-activated force without changes in the maximal ATPase activity and an increase in the Ca(2+) sensitivity of both ATPase and force development. No difference in the cross-bridge turnover rate was observed at the same level of cross-bridge attachment (activation state), showing that changes in Ca(2+) sensitivity were not due to changes in cross-bridge kinetics. Energy cost calculations demonstrated higher energy consumption in Tg-R145G fibers compared with Tg-WT fibers. The addition of 3 mm 2,3-butanedione monoxime at pCa 9.0 showed that there was approximately 2-4% of force generating cross-bridges attached in Tg-R145G fibers compared with less than 1.0% in Tg-WT fibers, suggesting that the mutation impairs the ability of the cardiac troponin complex to fully inhibit cross-bridge attachment under relaxing conditions. Prolonged force and intracellular [Ca(2+)] transients in electrically stimulated intact papillary muscles were observed in Tg-R145G compared with Tg-WT. These results suggest that the phenotype of hypertrophic cardiomyopathy is most likely caused by the compensatory mechanisms in the cardiovascular system that are activated by 1) higher energy cost in the heart resulting from a significant decrease in average force per cross-bridge, 2) slowed relaxation (diastolic dysfunction) caused by prolonged [Ca(2+)] and force transients, and 3) an inability of the cardiac TnI to completely inhibit activation in the absence of Ca(2+) in Tg-R145G mice.
Assuntos
Cardiomiopatia Hipertrófica/metabolismo , Troponina I/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Arginina/genética , Arginina/metabolismo , Peso Corporal , Cálcio/metabolismo , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/patologia , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Mutação/genética , Tamanho do Órgão , Troponina I/genéticaRESUMO
Restrictive cardiomyopathy (RCM) is a rare disorder characterized by impaired ventricular filling with decreased diastolic volume. We are reporting the functional effects of the first cardiac troponin T (CTnT) mutation linked to infantile RCM resulting from a de novo deletion mutation of glutamic acid 96. The mutation was introduced into adult and fetal isoforms of human cardiac TnT (HCTnT3-DeltaE96 and HCTnT1-DeltaE106, respectively) and studied with either cardiac troponin I (CTnI) or slow skeletal troponin I (SSTnI). Skinned cardiac fiber measurements showed a large leftward shift in the Ca(2+) sensitivity of force development with no differences in the maximal force. HCTnT1-DeltaE106 showed a significant increase in the activation of actomyosin ATPase with either CTnI or SSTnI, whereas HCTnT3-DeltaE96 was only able to increase the ATPase activity with CTnI. Both mutants showed an impaired ability to inhibit the ATPase activity. The capacity of the CTnI.CTnC and SSTnI.CTnC complexes to fully relax the fibers after TnT displacement was also compromised. Experiments performed using fetal troponin isoforms showed a less severe impact compared with the adult isoforms, which is consistent with the cardioprotective role of SSTnI and the rapid onset of RCM after birth following the isoform switch. These data indicate that troponin mutations related to RCM may have specific functional phenotypes, including large leftward shifts in the Ca(2+) sensitivity and impaired abilities to inhibit ATPase and to relax skinned fibers. All of this would account for and contribute to the severe diastolic dysfunction seen in RCM.
Assuntos
Sequência de Aminoácidos , Cálcio/metabolismo , Cardiomiopatia Restritiva/metabolismo , Cardiopatias Congênitas/metabolismo , Deleção de Sequência , Troponina T/metabolismo , Sequência de Aminoácidos/genética , Animais , Cálcio/química , Cardiomiopatia Restritiva/genética , Cardiopatias Congênitas/genética , Humanos , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/metabolismo , Relaxamento Muscular/genética , Miosinas/química , Miosinas/genética , Miosinas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Suínos , Troponina T/química , Troponina T/genética , Disfunção Ventricular/genética , Disfunção Ventricular/metabolismoRESUMO
The cytosolic Ca(2+)/Mg(2+)-binding protein alpha-parvalbumin (alpha-Parv) has been shown to accelerate cardiac relaxation; however, beyond an optimal concentration range, alpha-Parv can also diminish contractility. Mathematical modeling suggests that increasing Parv's Mg(2+) affinity may lower the effective concentration of Parv ([Parv]) to speed relaxation and, thus, limit Parv-mediated depressed contraction. Naturally occurring alpha/beta-Parv isoforms show divergence in amino acid primary structure (57% homology) and cation-binding affinities, with beta-Parv having an estimated 16% greater Mg(2+) affinity and approximately 200% greater Ca(2+) affinity than alpha-Parv. We tested the hypothesis that, at the same or lower estimated [Parv], mechanical relaxation rate would be more significantly accelerated by beta-Parv than by alpha-Parv. Dahl salt-sensitive (DS) rats were used as an experimental model of diastolic dysfunction. Relaxation properties were significantly slowed in adult cardiac myocytes isolated from DS rats compared with controls: time from peak contraction to 50% relaxation was 57 +/- 2 vs. 49 +/- 2 (SE) ms (P < 0.05), validating this model system. DS cardiac myocytes were subsequently transduced with alpha- or beta-Parv adenoviral vectors. Upon Parv gene transfer, beta-Parv caused significantly faster relaxation than alpha-Parv (P < 0.05), even though estimated [beta-Parv] was approximately 10% of [alpha-Parv]. This comparative analysis showing distinct functional outcomes raises the prospect of utilizing naturally occurring Parv variants to address disease-associated slowed cardiac relaxation.
Assuntos
Insuficiência Cardíaca/fisiopatologia , Hipertensão/fisiopatologia , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Parvalbuminas/metabolismo , Adenoviridae/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Progressão da Doença , Ecocardiografia , Técnicas de Transferência de Genes , Insuficiência Cardíaca/metabolismo , Hipertensão/metabolismo , Cinética , Masculino , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/fisiologia , Parvalbuminas/química , Parvalbuminas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Endogâmicos Dahl , Sarcômeros/fisiologiaRESUMO
Many biological systems use ethylene glycol bis (beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) to regulate the free calcium concentration ([Ca(2+)](free)) in the presence of physiological levels of free Mg(2+) ([Mg(2+)](free)). Frequently, it is necessary to work at [Ca(2+)](free) beyond EGTA's buffering capabilities. Therefore, we have developed methods to extend the buffering range by adding nitrilotriacetic acid (NTA) to solutions containing EGTA. This extension results from NTA having a lower K'(dCa) than EGTA. Such equilibria are solved by pCa Calculator, a computer program designed to aid in the study of Ca(2+)-dependent physiological processes while accounting for the effects of pH, temperature, and ionic strength. With multiple chelators and pH buffers from which to choose, pCa Calculator calculates the total concentration of each species required to achieve specified free concentrations of Ca(2+), ATP, and Mg(2+). The program is intuitive, user-friendly, and flexible enough to fix or vary the [Mg-ATP(2-)] and ionic strength. Moreover, it can account for increases in experimental volume from calcium addition. A comparative analysis is reported for testing solutions in the presence and absence of NTA by measuring the calcium binding affinity of fluorescent cardiac troponin C. These findings demonstrate that EGTA, when used in conjunction with NTA, improves and expands the regulation of free calcium in solution.
Assuntos
Cálcio/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação/genética , Quelantes/metabolismo , Técnicas de Química Analítica , Ácido Egtázico/metabolismo , Técnicas In Vitro , Cinética , Ligantes , Magnésio/metabolismo , Camundongos , Modelos Biológicos , Mutagênese Sítio-Dirigida , Ácido Nitrilotriacético/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Software , Design de Software , Soluções , Troponina C/química , Troponina C/genética , Troponina C/metabolismo , Interface Usuário-ComputadorRESUMO
We have studied the physiological effects of the troponin T (TnT) F110I and R278C mutations associated with familial hypertrophic cardiomyopathy (FHC) in humans. Three to four-month-old transgenic (Tg) mice expressing F110I-TnT and R278C-TnT did not develop significant hypertrophy or ventricular fibrosis even after chronic exercise challenge. The F110I mutation impaired acute exercise tolerance, whereas R278C did not. Skinned papillary muscle fibers from transgenic mice expressing F110I-TnT demonstrated increased Ca(2+) sensitivity of force and ATPase activity, and likewise an increased Ca(2+) sensitivity of force was observed in F110I-TnT-reconstituted human cardiac muscle preparations. In contrast, no changes in force or the ATPase-pCa dependencies were observed in transgenic R278C fibers or in human fibers reconstituted with the R278C-TnT mutant. The maximal level of force development was dramatically decreased in both transgenic mice. However, the maximal ATPase was not different (R278C-TnT) or only slightly less (F110I-TnT) than that of non-Tg and WT-Tg littermates. Consequently, their ratios of ATPase/force (energy cost) at all Ca(2+) concentrations were dramatically higher compared with non-Tg and WT-Tg fibers. This increase in energy cost most likely results from a decrease in force per myosin cross-bridge, because forcing all cross-bridges into the force generating state by substitution of MgADP for MgATP in maximum contracting solutions resulted in the same increase in maximal force (15%) in all transgenic and non-transgenic preparations. The combination of increased Ca(2+) sensitivity and energy cost in the F110I hearts may be responsible for the greater severity of this phenotype compared with the R278C mutation.
Assuntos
Cardiomiopatia Hipertrófica Familiar/genética , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Mutação/genética , Miocárdio/metabolismo , Troponina T/fisiologia , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Fibrose Endomiocárdica/metabolismo , Fibrose Endomiocárdica/patologia , Exercício Físico , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Natação , Troponina T/genéticaRESUMO
To study the functional consequences of various cardiomyopathic mutations in human cardiac alpha-tropomyosin (Tm), a method of depletion/reconstitution of native Tm and troponin (Tn) complex (Tm-Tn) in cardiac myofibril preparations has been developed. The endogenous Tm-Tn complex was selectively removed from myofibrils and replaced with recombinant wild-type or mutant proteins. Successful depletion and reconstitution steps were verified by SDS-gel electrophoresis and by the loss and regain of Ca2+-dependent regulation of ATPase activity. Five Tm mutations were chosen for this study: the hypertrophic cardiomyopathy (HCM) mutations E62Q, E180G, and L185R and the dilated cardiomyopathy (DCM) mutations E40K and E54K. Through the use of this new depletion/reconstitution method, the functional consequences of these mutations were determined utilizing myofibrillar ATPase measurements. The results of our studies showed that 1) depletion of >80% of Tm-Tn from myofibrils resulted in a complete loss of the Ca2+-regulated ATPase activity and a significant loss in the maximal ATPase activity, 2) reconstitution of exogenous wild-type Tm-Tn resulted in complete regain in the calcium regulation and in the maximal ATPase activity, and 3) all HCM-associated Tm mutations increased the Ca2+ sensitivity of ATPase activity and all had decreased abilities to inhibit ATPase activity. In contrast, the DCM-associated mutations both decreased the Ca2+ sensitivity of ATPase activity and had no effect on the inhibition of ATPase activity. These findings have demonstrated that the mutations which cause HCM and DCM disrupt discrete mechanisms, which may culminate in the distinct cardiomyopathic phenotypes.
Assuntos
Adenosina Trifosfatases/metabolismo , Cardiomiopatia Dilatada/genética , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Direita/genética , Tropomiosina/genética , Troponina/genética , Adenosina Trifosfatases/análise , Análise Mutacional de DNA , Engenharia Genética , Humanos , Miofibrilas/química , FenótipoRESUMO
The first human cardiac troponin I (hcTnI) mutation in the N-terminal 32 residue region, R21C (arginine residue number 21 mutated to cysteine), which has been linked to hypertrophic cardiomyopathy (HCM), has recently been reported. The effect of this mutation on the physiological function of hcTnI was investigated. Human cTnI R21C (in the absence or presence of troponin T and troponin C) was phosphorylated by protein kinase A (PKA) at a significantly slower rate than wild-type hcTnI. In skinned fiber studies, the TnI R21C mutant showed a large increase in Ca(2+)-sensitivity of force development when compared to wild-type TnI (DeltapCa(50)=0.33). Phosphorylation of skinned fibers containing TnI R21C by PKA resulted in a significantly smaller decrease in the Ca(2+)-sensitivity of force development when compared to phosphorylation of fibers containing wild-type TnI. The decreased sensitivity of TnI R21C to PKA is most likely due to a decreased ability of PKA to phosphorylate this TnI rather than conformational problems within this TnI. In addition, skinned fibers were found to contain an endogenous kinase that is capable of phosphorylating wild-type TnI. However, the endogenous kinase activity did not affect the Ca(2+)-sensitivity of force development, the Hill coefficient or maximal force of these skinned fibers. Actomyosin ATPase assays showed that the R21C mutation did not affect the inhibitory properties of TnI or the maximal ATPase activity. TnI R21C was also found to be more susceptible to proteolysis by calpain II than wild-type TnI. These results suggest that this R21C mutation in TnI affects the Ca(2+)-sensitizing effect of Tn, the ability of TnI to be readily phosphorylated by PKA and the stability of TnI to calpain. The results also suggest that the N-terminal region may have important roles such as modulating the Ca(2+)-sensitivity of force-development.
Assuntos
Cálcio/metabolismo , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Mutação/genética , Troponina I/genética , Troponina I/metabolismo , Actinas/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Cálcio/farmacologia , Calpaína/metabolismo , Dicroísmo Circular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Técnicas In Vitro , Miocárdio/metabolismo , Miosinas/antagonistas & inibidores , Miosinas/metabolismo , Fosforilação , Suínos , Fatores de Tempo , Troponina I/análise , Troponina I/químicaRESUMO
Human cardiac Troponin I (cTnI) is the first sarcomeric protein for which mutations have been associated with restrictive cardiomyopathy. To determine whether five mutations in cTnI (L144Q, R145W, A171T, K178E, and R192H) associated with restrictive cardiomyopathy were distinguishable from hypertrophic cardiomyopathy-causing mutations in cTnI, actomyosin ATPase activity and skinned fiber studies were carried out. All five mutations investigated showed an increase in the Ca2+ sensitivity of force development compared with wild-type cTnI. The two mutations with the worst clinical phenotype (K178E and R192H) both showed large increases in Ca2+ sensitivity (deltapCa50 = 0.47 and 0.36, respectively). Although at least one of these mutations is not in the known inhibitory regions of cTnI, all of the mutations investigated caused a decrease in the ability of cTnI to inhibit actomyosin ATPase activity. Mixtures of wild-type and mutant cTnI showed that cTnI mutants could be classified into three different groups: dominant (L144Q, A171T and R192H), equivalent (K178E), or weaker (R145W) than wild-type cTnI in actomyosin ATPase assays in the absence of Ca2+. Although most of the mutants were able to activate actomyosin ATPase similarly to wild-type cTnI, L144Q had significantly lower maximal ATPase activities than any of the other mutants or wild-type cTnI. Three mutants (L144Q, R145W, and K178E) were unable to fully relax contraction in the absence of Ca2+. The inability of the five cTnI mutations investigated to fully inhibit ATPase activity/force development and the generally larger increases in Ca2+ sensitivity than observed for most hypertrophic cardiomyopathy mutations would likely lead to severe diastolic dysfunction and may be the major physiological factors responsible for causing the restrictive cardiomyopathy phenotype in some of the genetically affected individuals.
Assuntos
Cálcio/metabolismo , Cardiomiopatia Restritiva , Contração Muscular/fisiologia , Mutação , Miocárdio/metabolismo , Miosinas/metabolismo , Troponina I , Animais , Cardiomiopatia Restritiva/genética , Cardiomiopatia Restritiva/metabolismo , Humanos , Técnicas In Vitro , Modelos Moleculares , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Miocárdio/citologia , Estrutura Terciária de Proteína , Suínos , Troponina/química , Troponina/metabolismo , Troponina I/genética , Troponina I/metabolismoRESUMO
We have proposed that pharmacological preconditioning, leading to PKC-epsilon activation, in hearts improves postischemic functional recovery through a decrease in actomyosin ATPase activity and subsequent ATP conservation. The purpose of the present study was to determine whether moderate PKC-independent decreases in actomyosin ATPase are sufficient to improve myocardial postischemic function. Rats were given propylthiouracil (PTU) for 8 days to induce a 25% increase in beta-myosin heavy chain with a 28% reduction in actomyosin ATPase activity. Recovery of postischemic left ventricular developed pressure (LVDP) was significantly higher in PTU-treated rat hearts subjected to 30 min of global ischemia than in control hearts: 57.9 +/- 6.2 vs. 32.6 +/- 5.1% of preischemic values. In addition, PTU-treated hearts exhibited a delayed onset of rigor contracture during ischemia and a higher global ATP content after ischemia. In the second part of our study, we demonstrated a lower maximal actomyosin ATPase and a higher global ATP content after ischemia in human troponin T (TnT) transgenic mouse hearts. In mouse hearts with and without a point mutation at F110I of human TnT, recovery of postischemic LVDP was 55.4 +/- 5.5 and 62.5 +/- 14.5% compared with 20.0 +/- 2.9% in nontransgenic mouse hearts after 35 min of global ischemia. These results are consistent with the hypothesis that moderate decreases in actomyosin ATPase activity result in net ATP conservation that is sufficient to improve postischemic contractile function.
Assuntos
Actomiosina/metabolismo , Metabolismo Energético/fisiologia , Isquemia Miocárdica/metabolismo , Miocárdio/metabolismo , Adenina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Creatina/metabolismo , Feminino , Humanos , Hipotireoidismo/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Cadeias Pesadas de Miosina/metabolismo , Proteína Quinase C/metabolismo , Proteína Quinase C-delta , Proteína Quinase C-épsilon , Ratos , Ratos Sprague-Dawley , Troponina T/genética , Troponina T/metabolismoRESUMO
In this study we investigated the physiological role of the cardiac troponin T (cTnT) isoforms in the presence of human slow skeletal troponin I (ssTnI). ssTnI is the main troponin I isoform in the fetal human heart. In reconstituted fibers containing the cTnT isoforms in the presence of ssTnI, cTnT1-containing fibers showed increased Ca(2+) sensitivity of force development compared with cTnT3- and cTnT4-containing fibers. The maximal force in reconstituted skinned fibers was significantly greater for the cTnT1 (predominant fetal cTnT isoform) when compared with cTnT3 (adult TnT isoform) in the presence of ssTnI. Troponin (Tn) complexes containing ssTnI and reconstituted with cTnT isoforms all yielded different maximal actomyosin ATPase activities. Tn complexes containing cTnT1 and cTnT4 (both fetal isoforms) had a reduced ability to inhibit actomyosin ATPase activity when compared with cTnT3 (adult isoform) in the presence of ssTnI. The rate at which Ca(2+) was released from site II of cTnC in the cTnI.cTnC complex (122/s) was 12.5-fold faster than for the ssTnI.cTnC complex (9.8/s). Addition of cTnT3 to the cTnI.cTnC complex resulted in a 3.6-fold decrease in the Ca(2+) dissociation rate from site II of cTnC. Addition of cTnT3 to the ssTnI.cTnC complex resulted in a 1.9-fold increase in the Ca(2+) dissociation rate from site II of cTnC. The rate at which Ca(2+) dissociated from site II of cTnC in Tn complexes also depended on the cTnT isoform present. However, the TnI isoforms had greater effects on the Ca(2+) dissociation rate of site II than the cTnT isoforms. These results suggest that the different N-terminal TnT isoforms would produce distinct functional properties in the presence of ssTnI when compared with cTnI and that each isoform would have a specific physiological role in cardiac muscle.
Assuntos
Cálcio/metabolismo , Troponina I/metabolismo , Troponina T/metabolismo , Humanos , Cinética , Miosinas/metabolismo , Isoformas de Proteínas/metabolismo , Fatores de TempoRESUMO
The effects of Troponin T (TnT) mutants R141W and DeltaK210, the only two currently known mutations in TnT that cause dilated cardiomyopathy(DCM) independent of familial hypertrophic cardiomyopathy (FHC), and TnT-K273E, a mutation that leads to a progression from FHC to DCM, were investigated. Studies on the Ca2+ sensitivity of force development in porcine cardiac fibers demonstrated that TnT-DeltaK210 caused a significant decrease in Ca2+ sensitivity, whereas the TnT-R141W did not result in any change in Ca2+ sensitivity when compared with human cardiac wild-type TnT (HCWTnT). TnT-DeltaK210 also caused a decrease in maximal force when compared with HCWTnT and TnT-R141W. In addition, the TnT-DeltaK210 mutant decreased maximal ATPase activity in the presence of Ca2+. However, the TnT-K273E mutation caused a significant increase in Ca2+ sensitivity but behaved similarly to HCWTnT in actomyosin activation assays. Inhibition of ATPase activity in reconstituted actin-activated myosin ATPase assays was similar for all three TnT mutants and HCWTnT. Additionally, circular dichroism studies suggest that the secondary structure of all three TnT mutants was similar to that of the HCWTnT. These results suggest that a rightward shift in Ca2+ sensitivity is not the only determinant for the phenotype of DCM.