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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 Ca2+ binding sensitivity of the thin filament.Panel A shows the Ca2+ dependent increases in IAANS fluorescence for thin filament bound control  (□), and the DCM mutants, TnI K36Q  (), TnT R131W (○), TnT R141W  (•), TnT R205L  (▪) and TnT ΔK210  (⋆) as a function of pCa. Panel B shows the Ca2+ dependent increases in IAANS fluorescence for thin filament bound control  (□), and the HCM mutants, TnT R92Q  (▾) and TnI S166F  (▴) as a function of pCa. Panel C shows the Ca2+ dependent increases in IAANS fluorescence for thin filament bound control  (□), and the RCM mutants, TnI D190H  (▽) and TnI R192H  (▵) as a function of pCa. Panel D shows the Ca2+ dependent increases in IAANS fluorescence for thin filament bound control  (□) and ischemic related truncated TnI (1-192)  () as a function of pCa. The data sets were normalized individually for each mutant. 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. The Ca2+ sensitivities were reported as a dissociation constant Kd, representing a mean of three to four separate titrations ± S.E.M.
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pone-0038259-g003: Effect of disease-related protein modifications on the Ca2+ binding sensitivity of the thin filament.Panel A shows the Ca2+ dependent increases in IAANS fluorescence for thin filament bound control (□), and the DCM mutants, TnI K36Q (), TnT R131W (○), TnT R141W (•), TnT R205L (▪) and TnT ΔK210 (⋆) as a function of pCa. Panel B shows the Ca2+ dependent increases in IAANS fluorescence for thin filament bound control (□), and the HCM mutants, TnT R92Q (▾) and TnI S166F (▴) as a function of pCa. Panel C shows the Ca2+ dependent increases in IAANS fluorescence for thin filament bound control (□), and the RCM mutants, TnI D190H (▽) and TnI R192H (▵) as a function of pCa. Panel D shows the Ca2+ dependent increases in IAANS fluorescence for thin filament bound control (□) and ischemic related truncated TnI (1-192) () as a function of pCa. The data sets were normalized individually for each mutant. 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. The Ca2+ sensitivities were reported as a dissociation constant Kd, representing a mean of three to four separate titrations ± S.E.M.

Mentions: Thin filament bound control exhibited a Ca2+-dependent half-maximal fluorescence increase at 4.5±0.2 µM (Figure 3 and Table 2). Consistent with the general Ca2+ desensitizing effects of DCM mutations [5], [13], all five DCM mutations studied here desensitized Ca2+ binding to the thin filament. TnT R131W, R141W and R205L slightly desensitized Ca2+ binding to the thin filament by ∼ 1.5 fold; TnI K36Q had an intermediate Ca2+ desensitizing effect of ∼ 2-fold; while TnT ΔK210 had the largest Ca2+ desensitizing effect of ∼ 3.5-fold (Figure 3C and Table 2). Of the Ca2+ desensitizing modifications, only TnT ΔK210 significantly decreased the Ca2+ binding cooperativity of the thin filament by ∼ 1.3-fold (Figure 3C and Table 2).


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 Ca2+ binding sensitivity of the thin filament.Panel A shows the Ca2+ dependent increases in IAANS fluorescence for thin filament bound control  (□), and the DCM mutants, TnI K36Q  (), TnT R131W (○), TnT R141W  (•), TnT R205L  (▪) and TnT ΔK210  (⋆) as a function of pCa. Panel B shows the Ca2+ dependent increases in IAANS fluorescence for thin filament bound control  (□), and the HCM mutants, TnT R92Q  (▾) and TnI S166F  (▴) as a function of pCa. Panel C shows the Ca2+ dependent increases in IAANS fluorescence for thin filament bound control  (□), and the RCM mutants, TnI D190H  (▽) and TnI R192H  (▵) as a function of pCa. Panel D shows the Ca2+ dependent increases in IAANS fluorescence for thin filament bound control  (□) and ischemic related truncated TnI (1-192)  () as a function of pCa. The data sets were normalized individually for each mutant. 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. The Ca2+ sensitivities were reported as a dissociation constant Kd, representing a mean of three to four separate titrations ± S.E.M.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366952&req=5

pone-0038259-g003: Effect of disease-related protein modifications on the Ca2+ binding sensitivity of the thin filament.Panel A shows the Ca2+ dependent increases in IAANS fluorescence for thin filament bound control (□), and the DCM mutants, TnI K36Q (), TnT R131W (○), TnT R141W (•), TnT R205L (▪) and TnT ΔK210 (⋆) as a function of pCa. Panel B shows the Ca2+ dependent increases in IAANS fluorescence for thin filament bound control (□), and the HCM mutants, TnT R92Q (▾) and TnI S166F (▴) as a function of pCa. Panel C shows the Ca2+ dependent increases in IAANS fluorescence for thin filament bound control (□), and the RCM mutants, TnI D190H (▽) and TnI R192H (▵) as a function of pCa. Panel D shows the Ca2+ dependent increases in IAANS fluorescence for thin filament bound control (□) and ischemic related truncated TnI (1-192) () as a function of pCa. The data sets were normalized individually for each mutant. 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. The Ca2+ sensitivities were reported as a dissociation constant Kd, representing a mean of three to four separate titrations ± S.E.M.
Mentions: Thin filament bound control exhibited a Ca2+-dependent half-maximal fluorescence increase at 4.5±0.2 µM (Figure 3 and Table 2). Consistent with the general Ca2+ desensitizing effects of DCM mutations [5], [13], all five DCM mutations studied here desensitized Ca2+ binding to the thin filament. TnT R131W, R141W and R205L slightly desensitized Ca2+ binding to the thin filament by ∼ 1.5 fold; TnI K36Q had an intermediate Ca2+ desensitizing effect of ∼ 2-fold; while TnT ΔK210 had the largest Ca2+ desensitizing effect of ∼ 3.5-fold (Figure 3C and Table 2). Of the Ca2+ desensitizing modifications, only TnT ΔK210 significantly decreased the Ca2+ binding cooperativity of the thin filament by ∼ 1.3-fold (Figure 3C and Table 2).

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