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Disease-related cardiac troponins alter thin filament Ca2+ association and dissociation rates.

Liu B, Tikunova SB, Kline KP, Siddiqui JK, Davis JP - PLoS ONE (2012)

Bottom Line: By utilizing an IAANS labeled fluorescent troponin C, [Formula: see text], we examined the effects of ten disease-related troponin modifications on the Ca(2+) binding properties of the troponin complex and the reconstituted thin filament.Consistent with previous studies, the majority of the protein modifications had no effect on the Ca(2+) binding properties of the isolated troponin complex.The protein modifications also increased (up to 5.4-fold) or decreased (up to 2.5-fold) the apparent rate of Ca(2+) association to the thin filament.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Cell Biology, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
The contractile response of the heart can be altered by disease-related protein modifications to numerous contractile proteins. By utilizing an IAANS labeled fluorescent troponin C, [Formula: see text], we examined the effects of ten disease-related troponin modifications on the Ca(2+) binding properties of the troponin complex and the reconstituted thin filament. The selected modifications are associated with a broad range of cardiac diseases: three subtypes of familial cardiomyopathies (dilated, hypertrophic and restrictive) and ischemia-reperfusion injury. Consistent with previous studies, the majority of the protein modifications had no effect on the Ca(2+) binding properties of the isolated troponin complex. However, when incorporated into the thin filament, dilated cardiomyopathy mutations desensitized (up to 3.3-fold), while hypertrophic and restrictive cardiomyopathy mutations, and ischemia-induced truncation of troponin I, sensitized the thin filament to Ca(2+) (up to 6.3-fold). Kinetically, the dilated cardiomyopathy mutations increased the rate of Ca(2+) dissociation from the thin filament (up to 2.5-fold), while the hypertrophic and restrictive cardiomyopathy mutations, and the ischemia-induced truncation of troponin I decreased the rate (up to 2-fold). The protein modifications also increased (up to 5.4-fold) or decreased (up to 2.5-fold) the apparent rate of Ca(2+) association to the thin filament. Thus, the disease-related protein modifications alter Ca(2+) binding by influencing both the association and dissociation rates of thin filament Ca(2+) exchange. These alterations in Ca(2+) exchange kinetics influenced the response of the thin filament to artificial Ca(2+) transients generated in a stopped-flow apparatus. Troponin C may act as a hub, sensing physiological and pathological stimuli to modulate the Ca(2+)-binding properties of the thin filament and influence the contractile performance of the heart.

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Effect of disease-related protein modifications on the rate of Ca2+ dissociation from the Tn complex.Panel A shows the time courses of the increase in IAANS fluorescence as Ca2+ was removed by EGTA from control , and the DCM mutants, TnI K36Q , TnT R131W , TnT R141W , TnT R205L  and TnT ΔK210 . Panel B shows the time courses of the increase in IAANS fluorescence as Ca2+ was removed by EGTA from control , and the HCM mutants, TnT R92Q  and TnI S166F . Panel C shows the time courses of the increase in IAANS fluorescence as Ca2+ was removed by EGTA from control , and the RCM mutants, TnI D190H  and TnI R192H . Panel D shows the time courses of the increase in IAANS fluorescence as Ca2+ was removed by EGTA from control  and ischemic related truncated TnI (1-192) . All  complexes consist of the full length Tn subunits of , TnI and TnT, except for ischemic related truncated TnI (1-192). The disease related modification is either in TnI or TnT, in either case, the other protein (TnT or TnI) was wild type. Data traces (an average of 3 to 5 individual traces collected at least 10 times) were fit with a single exponential equation to calculate the kinetic rates. The data traces have been staggered and normalized for clarity.
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pone-0038259-g002: Effect of disease-related protein modifications on the rate of Ca2+ dissociation from the Tn complex.Panel A shows the time courses of the increase in IAANS fluorescence as Ca2+ was removed by EGTA from control , and the DCM mutants, TnI K36Q , TnT R131W , TnT R141W , TnT R205L and TnT ΔK210 . Panel B shows the time courses of the increase in IAANS fluorescence as Ca2+ was removed by EGTA from control , and the HCM mutants, TnT R92Q and TnI S166F . Panel C shows the time courses of the increase in IAANS fluorescence as Ca2+ was removed by EGTA from control , and the RCM mutants, TnI D190H and TnI R192H . Panel D shows the time courses of the increase in IAANS fluorescence as Ca2+ was removed by EGTA from control and ischemic related truncated TnI (1-192) . All complexes consist of the full length Tn subunits of , TnI and TnT, except for ischemic related truncated TnI (1-192). The disease related modification is either in TnI or TnT, in either case, the other protein (TnT or TnI) was wild type. Data traces (an average of 3 to 5 individual traces collected at least 10 times) were fit with a single exponential equation to calculate the kinetic rates. The data traces have been staggered and normalized for clarity.

Mentions: Previously, we demonstrated that the fluorescence of reported the actual rate of Ca2+ dissociation from unlabeled wild type Tn and a series of rationally engineered mutant Tn complexes with high fidelity [11], [25]. In this study, fluorescence stopped-flow measurements were performed to determine the rate of Ca2+ dissociation from the disease-related complexes. Figure 2 shows that the rate of Ca2+ dissociation from control was 40.8±0.4/s (Table 1). Consistent with its effect on the Ca2+ sensitivity of Tn, HCM TnI S166F slowed the rate of Ca2+ dissociation ∼2-fold (Figure 2 and Table 1). Besides TnI S166F, only TnI K36Q slightly, but significantly altered the rate of Ca2+ dissociation from the Tn complex (∼ 1.2-fold faster; Figure 2A and Table 1).


Disease-related cardiac troponins alter thin filament Ca2+ association and dissociation rates.

Liu B, Tikunova SB, Kline KP, Siddiqui JK, Davis JP - PLoS ONE (2012)

Effect of disease-related protein modifications on the rate of Ca2+ dissociation from the Tn complex.Panel A shows the time courses of the increase in IAANS fluorescence as Ca2+ was removed by EGTA from control , and the DCM mutants, TnI K36Q , TnT R131W , TnT R141W , TnT R205L  and TnT ΔK210 . Panel B shows the time courses of the increase in IAANS fluorescence as Ca2+ was removed by EGTA from control , and the HCM mutants, TnT R92Q  and TnI S166F . Panel C shows the time courses of the increase in IAANS fluorescence as Ca2+ was removed by EGTA from control , and the RCM mutants, TnI D190H  and TnI R192H . Panel D shows the time courses of the increase in IAANS fluorescence as Ca2+ was removed by EGTA from control  and ischemic related truncated TnI (1-192) . All  complexes consist of the full length Tn subunits of , TnI and TnT, except for ischemic related truncated TnI (1-192). The disease related modification is either in TnI or TnT, in either case, the other protein (TnT or TnI) was wild type. Data traces (an average of 3 to 5 individual traces collected at least 10 times) were fit with a single exponential equation to calculate the kinetic rates. The data traces have been staggered and normalized for clarity.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366952&req=5

pone-0038259-g002: Effect of disease-related protein modifications on the rate of Ca2+ dissociation from the Tn complex.Panel A shows the time courses of the increase in IAANS fluorescence as Ca2+ was removed by EGTA from control , and the DCM mutants, TnI K36Q , TnT R131W , TnT R141W , TnT R205L and TnT ΔK210 . Panel B shows the time courses of the increase in IAANS fluorescence as Ca2+ was removed by EGTA from control , and the HCM mutants, TnT R92Q and TnI S166F . Panel C shows the time courses of the increase in IAANS fluorescence as Ca2+ was removed by EGTA from control , and the RCM mutants, TnI D190H and TnI R192H . Panel D shows the time courses of the increase in IAANS fluorescence as Ca2+ was removed by EGTA from control and ischemic related truncated TnI (1-192) . All complexes consist of the full length Tn subunits of , TnI and TnT, except for ischemic related truncated TnI (1-192). The disease related modification is either in TnI or TnT, in either case, the other protein (TnT or TnI) was wild type. Data traces (an average of 3 to 5 individual traces collected at least 10 times) were fit with a single exponential equation to calculate the kinetic rates. The data traces have been staggered and normalized for clarity.
Mentions: Previously, we demonstrated that the fluorescence of reported the actual rate of Ca2+ dissociation from unlabeled wild type Tn and a series of rationally engineered mutant Tn complexes with high fidelity [11], [25]. In this study, fluorescence stopped-flow measurements were performed to determine the rate of Ca2+ dissociation from the disease-related complexes. Figure 2 shows that the rate of Ca2+ dissociation from control was 40.8±0.4/s (Table 1). Consistent with its effect on the Ca2+ sensitivity of Tn, HCM TnI S166F slowed the rate of Ca2+ dissociation ∼2-fold (Figure 2 and Table 1). Besides TnI S166F, only TnI K36Q slightly, but significantly altered the rate of Ca2+ dissociation from the Tn complex (∼ 1.2-fold faster; Figure 2A and Table 1).

Bottom Line: By utilizing an IAANS labeled fluorescent troponin C, [Formula: see text], we examined the effects of ten disease-related troponin modifications on the Ca(2+) binding properties of the troponin complex and the reconstituted thin filament.Consistent with previous studies, the majority of the protein modifications had no effect on the Ca(2+) binding properties of the isolated troponin complex.The protein modifications also increased (up to 5.4-fold) or decreased (up to 2.5-fold) the apparent rate of Ca(2+) association to the thin filament.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Cell Biology, The Ohio State University, Columbus, Ohio, United States of America.

ABSTRACT
The contractile response of the heart can be altered by disease-related protein modifications to numerous contractile proteins. By utilizing an IAANS labeled fluorescent troponin C, [Formula: see text], we examined the effects of ten disease-related troponin modifications on the Ca(2+) binding properties of the troponin complex and the reconstituted thin filament. The selected modifications are associated with a broad range of cardiac diseases: three subtypes of familial cardiomyopathies (dilated, hypertrophic and restrictive) and ischemia-reperfusion injury. Consistent with previous studies, the majority of the protein modifications had no effect on the Ca(2+) binding properties of the isolated troponin complex. However, when incorporated into the thin filament, dilated cardiomyopathy mutations desensitized (up to 3.3-fold), while hypertrophic and restrictive cardiomyopathy mutations, and ischemia-induced truncation of troponin I, sensitized the thin filament to Ca(2+) (up to 6.3-fold). Kinetically, the dilated cardiomyopathy mutations increased the rate of Ca(2+) dissociation from the thin filament (up to 2.5-fold), while the hypertrophic and restrictive cardiomyopathy mutations, and the ischemia-induced truncation of troponin I decreased the rate (up to 2-fold). The protein modifications also increased (up to 5.4-fold) or decreased (up to 2.5-fold) the apparent rate of Ca(2+) association to the thin filament. Thus, the disease-related protein modifications alter Ca(2+) binding by influencing both the association and dissociation rates of thin filament Ca(2+) exchange. These alterations in Ca(2+) exchange kinetics influenced the response of the thin filament to artificial Ca(2+) transients generated in a stopped-flow apparatus. Troponin C may act as a hub, sensing physiological and pathological stimuli to modulate the Ca(2+)-binding properties of the thin filament and influence the contractile performance of the heart.

Show MeSH
Related in: MedlinePlus