A σ-bonded cobalt allene

123

(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 proteinprotein interactions along the sarcomere lattice disrupting the coupling between the cross-bridge and Ca 2؉ binding to cTnC.Hypertrophic cardiomyopathy (HCM) 3 is typically inherited as an autosomal dominant disease that is caused by mutations in sarcomeric genes and is the most prevalent cause of sudden death in athletes and young people (1, 2). The clinical hallmark of HCM is an increased thickness of the left ventricular wall. Myocyte disarray, fibrosis, septal hypertrophy, and abnormal diastolic function can also be present in HCM patients (3). HCM mutations have been reported in 13 myofilament-related genes; however, the cardiac troponin C (cTnC) gene remained excluded from this list (4 -7). The clinical and functional phenotypes may vary according to the gene and the location of the mutation (8). Recently our group has reported evidence that brings cTnC into focus as an HCM susceptibility gene (9). Interestingly the prevalence for cTnC HCM mutations was the same as other well characterized genes (i.e. actin and tropomyosin) (6). To date, prior to our recent report, only one mutation in cTnC (L29Q) had been linked to HCM (10). In vitro and in situ studies demonstrating changes in the functional parameters of cardiac muscle regulation suggest that this mutation is causative...

Section: Actomyosin Atpase Pca Curves Using Reconstituted Troponin Comentioning

confidence: 99%

“…The data were fitted to a version of the Hill equation that accounted for the spectral changes that occur at a low Ca 2ϩ concentration and plotted using SigmaPlot 10.0. The apparent Ca 2ϩ affinities are reported as pCa 50 values ϮS.E.…”

Section: Determination Of Apparent Ca 2؉ Affinities By Fluorescencementioning

confidence: 99%

A Functional and Structural Study of Troponin C Mutations Related to Hypertrophic Cardiomyopathy

Pinto

Parvatiyar

Jones

et al. 2009

123

“…Several groups (24,(41)(42)(43)(44) have demonstrated that the binding of Ca 2ϩ to the low affinity site initiates muscle contraction. Removal of Ca 2ϩ from the Ca 2ϩ -specific sites in skeletal TnC has also been linked to structural changes in TnC (41,45,46). The effect of cTnT and/or TnI isoforms on the rate of Ca 2ϩ removal from either the high or low affinity Ca 2ϩ -binding sites on cTnC has not been examined previously.…”

Section: ϫ015) Than Intact Fibers Similar To What Has Been Reported mentioning

confidence: 99%

Cardiac Troponin T Isoforms Affect the Ca2+ Sensitivity of Force Development in the Presence of Slow Skeletal Troponin I

Gomes¹,

Venkatraman

Davis

et al. 2004

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 cTnI⅐cTnC complex (122/s) was 12.5-fold faster than for the ssTnI⅐cTnC complex (9.8/s). Addition of cTnT3 to the cTnI⅐cTnC complex resulted in a 3.6-fold decrease in the Ca 2؉ dissociation rate from site II of cTnC. Addition of cTnT3 to the ssTnI⅐cTnC 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.

“…Previous studies in this laboratory (49) have shown that of the total Ca 2ϩ -induced ellipticity in intact TnC, ϳ27% could be assigned to N domain and ϳ73% to C domain. Rather similar large contributions by C domain were reported by Johnson and Potter (50). To eliminate the large background of -activated tensions (pCa 6.2-4.4) were measured in native rabbit psoas fibers, followed by extraction of endogenous TnC and reconstitution with sTnC F29W or chicken muscle TnC, as described under "Materials and Methods."…”

Section: Tension Measurements-mentioning

confidence: 99%

Biological Function and Site II Ca2+-induced Opening of the Regulatory Domain of Skeletal Troponin C Are Impaired by Invariant Site I or II Glu Mutations

Pearlstone¹,

Chandra²,

Sorenson³

et al. 2000