Reciprocal coupling between troponin C and myosin crossbridge attachment

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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 ϳ23% 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.

Section: Discussionsupporting

confidence: 66%

A Dilated Cardiomyopathy Troponin C Mutation Lowers Contractile Force by Reducing Strong Myosin-Actin Binding

Dweck¹,

Reynaldo

Pinto³

et al. 2010

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“…These factors would directly impact the amount of force or degree of activation that is achieved. One result of decreased cross-bridge formation is a diminished cTnC Ca 2ϩ affinity because these two processes are tightly coupled (62).…”

Section: Discussionmentioning

confidence: 99%

Predicting Cardiomyopathic Phenotypes by Altering Ca2+ Affinity of Cardiac Troponin C

Parvatiyar

Pinto

Liang

et al. 2010

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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 ␣-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.

“…k off(TnC⅐Ca) therefore becomes smaller as cross-bridges form and force develops (34,37,38). The exponential dependence of k off(TnC⅐Ca) on force is based on experimental observations in cardiac and skeletal muscle (32)(33)(34) and is quantified by the equation k off.TnC⅐Ca ϭ k off.TnC⅐Ca.rest ϫ e Ϫgain ϫ relative force , where k off.TnC⅐Ca.rest is the off-rate of Ca 2ϩ from TnC in the absence of force development.…”

Section: Force Dependence Of Affinity Of Tnc For Ca 2ϩmentioning

confidence: 99%

Functional Analysis of a Troponin I (R145G) Mutation Associated with Familial Hypertrophic Cardiomyopathy

Lang¹,

Gomes²,

Zhao³

et al. 2002

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Familial hypertrophic cardiomyopathy has been associated with several mutations in the gene encoding human cardiac troponin I (HCTnI). A missense mutation in the inhibitory region of TnI replaces an arginine residue at position 145 with a glycine and cosegregates with the disease. Results from several assays indicate that the inhibitory function of HCTnI R145G is significantly reduced. When HCTnI R145G was incorporated into whole troponin, Tn R145G (HCTnT⅐HCTnI R145G ⅐HCTnC), only partial inhibition of the actin-tropomyosin-myosin ATPase activity was observed in the absence of Ca 2؉ compared with wild type Tn (HCTnT⅐HCTnI⅐HCTnC). Maximal activation of actin-tropomyosin-myosin ATPase in the presence of Ca 2؉ was also decreased in Tn R145G when compared with Tn. Using skinned cardiac muscle fibers, we determined that in comparison with the wild type complex 1) the complex containing HCTnI R145G only inhibited 84% of Ca 2؉ -unregulated force, 2) the recovery of Ca 2؉ -activated force was decreased, and 3) there was a significant increase in the Ca 2؉ sensitivity of force development. Computer modeling of troponin C and I variables predicts that the primary defect in TnI caused by these mutations would lead to diastolic dysfunction. These results suggest that severe diastolic dysfunction and somewhat decreased contractility would be prominent clinical features and that hypertrophy could arise as a compensatory mechanism. Familial hypertrophic cardiomyopathy (FHC)1 has been linked to mutations in genes of nine different sarcomeric proteins. These mutations have been found in the genes for ␣-myosin heavy chain (1), cardiac myosin essential light chain and cardiac myosin regulatory light chain (2), ␣-tropomyosin, cardiac troponin T (TnT) (3), cardiac myosin-binding protein C (4, 5), troponin I (TnI) (6), ␣-actin (7), as well as titin (8), and possibly troponin C (TnC) (9). This disease has recently gained significant attention due to several highly publicized reports of sudden death and fainting spells in young athletes who were asymptomatic and otherwise healthy individuals. In general, patients with FHC demonstrate an increase in heart muscle mass and sometimes an irregular echocardiogram (10). Kimura et al. (6) reported five missense mutations in TnI, R145G, R145Q, R162W, G203S, and K206Q, that cosegregate with FHC (Fig. 1). Three other TnI FHC mutants (S199N, Lys-183 deletion, and an exon 8 deletion mutant encompassing the stop codon of the cardiac TnI gene) have recently been discovered (11, 12). Functionally TnI is the inhibitory subunit of the troponin (Tn) complex that controls the interaction between actin and myosin in a Ca 2ϩ -dependent manner (13-15). Studies using proteolytic fragments of fast skeletal TnI identified the central TnI sequence (residues 96 -116) as being responsible for its inhibitory activity. Residues 104 -115 of fast skeletal TnI (comparable to residues 136 -147 in cardiac TnI) formed the minimum sequence necessary for inhibition of muscle contraction (16 -19). Two of these mutations occ...